JP2004502782A - Multiple antigenic peptides that are immunogenic against Streptococcus pneumoniae - Google Patents

Abstract

The present invention relates to S.I. A multi-antigenic peptide [(multiple antigenic peptide) (multiple antigenic peptide)] (MAP) that confers protection against pneumoniae challenge is provided. These multi-antigenic peptides include peptides that immunospecifically bind to monoclonal antibodies. Also provided is a method of conferring protective immunity against Streptococcus pneumoniae by administering a vaccine comprising such an immunogenic peptide and a therapeutic composition comprising the MAP of the present invention.

Description

^ａ全ての２３のワクチン型の各々２〜３の株の調査を含む最初のＴｓｐ５０９Ｉ分析
^ｂさらなるＴｓｐ５０９Ｉ部位を示す型のさらなる株のＴｓｐ５０９Ｉ分析
^ｃ括弧に示されるものは、さらなる制限部位を有する血清型の数と試験した血清型の数の比である。
【０１１９】
この分析は、Ｓ．ｐｎｅｕｍｏｎｉａｅ血清型６Ｂ由来のＰｓａＡをコードする遺伝子のクローニングおよび配列決定ならびに、２３種の肺炎球菌ポリサッカライドワクチン血清型における引き続く遺伝子の分析を開示する。配列分析によって、血清型６Ｂ配列および以前に公開された株Ｒ３６Ａが、予想よりも類似していないことが明らかになった。ヌクレオチド配列およびその隣接領域は、起源の株Ｒ３６Ａ　ＰｓａＡに対して７３％のみに相同であったが、実際のＰｓａＡコード配列は、７８％の計算された相同性を有した。２つの配列間のタンパク質配列類似性は、８１％しかなかった。新しく提供された血清型２肺炎球菌ＰｓａＡ（ワクチン血清型）と血清型６Ｂ配列の比較によって、ＤＮＡ相同性は９９％および９８％タンパク質配列類似性と計算された。さらに、これらの値は、ワクチン血清型内の遺伝子についての配列保存が、高度である証拠である。これらの２つの配列の推定アミノ酸配列を、属内のＰｓａＡ相同性について他の公開された配列と比較した場合、類似性の大きな領域は、全ての５種のタンパク質に明白であった。群内の類似性値は、５７〜８２％の範囲であった。
【０１２０】
Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｐｎｅｕｍｏｎｉａｅワクチン候補の必要性は、本発明者らが、Ｓ．ｐｎｅｕｍｏｎｉａｅ血清型６ＢからＰｓａＡ遺伝子を、クローニングし、そして配列決定することを促進した。２つの肺炎球菌ＰｓａＡ遺伝子（６ＢおよびＲ３６Ａ）の間の不均質性によって、本発明者らは、ワクチン血清型を試験し、株間の多様性の程度を決定した。Ｓ．ｐｎｅｕｍｏｎｉａｅ血清型６ＢからのＰｓａＡ遺伝子のＮ末端コード領域およびＣ末端コード領域に対応するプライマーは、２３個全てのワクチン血清型を増幅した。血清型６Ｂ遺伝子のうちに２１個の部位を表す１０個の異なる制限酵素を使用するＰＣＲ−ＲＦＬＰ分析は、株間でたった１つの多様性領域を示しただけであり、これは、少数の株についてさらなるＴｓｐ５０９Ｉ部位を生じた。本研究は、血清型６Ｂ遺伝子の配列は、ワクチン血清型の間で見出される配列を代表することを実証する。このことについての証拠としては、血清型２と血清型６Ｂとの間の９９％のＤＮＡ配列同一性、および本研究において試験された２１個の部位を網羅する均一かつ同一な制限パターンが挙げられる。より初期の株Ｒ３６Ａ　ＰｓａＡ配列は、本明細書で分析された血清型において表面上は存在しない改変体配列を表すことが明らかである。なぜなら、発明者らは、株Ｒ３６Ａ　ＰｓａＡに対してプライマーを使用して、この配列を増幅することが不可能だったからである。しかし、本研究のより重要な局面は、分析したワクチン血清型間で、限定された多様性が存在することである。疾患を引き起こし、従ってそれに対する予防的手段が必要とされるものは、血清型である。これらの血清型の間でのＰｓａＡの遺伝的多様性の欠損は、遺伝子が高度に保存されており、そしてワクチン開発のための優れた候補であることを示唆する。
【０１２１】
（実施例９．モノクローナル抗体）
血清型２２Ｆ由来の３７ｋＤａのタンパク質を使用して、モノクローナル抗体（１Ｂ６Ｅ１２Ｈ９、３Ｃ４Ｄ５Ｃ７、４Ｅ９Ｇ９Ｄ３、４Ｈ５Ｃ１０Ｆ３、６Ｆ６Ｆ９Ｃ８、および８Ｇ１２Ｇ１１Ｂ１０）を作製した（米国特許出願番号０８／７１５，１３１、現在は米国特許５，８５４，４１６号（これらは参考として本明細書中に援用される）において開示される）。これらのＭＡｂを、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｐｎｅｕｍｏｎｉａｅによる感染からの防御を付与するその能力について、分析した。表２は、試験した５つのモノクローナル抗体の能力を示し、特に１つが、その後のＳ．ｐｎｅｕｍｏｎｉａｅチャレンジからの効率的な防御を与えた（８Ｇ１２Ｇ１１Ｂ１０）。Ｓ．ｐｎｅｕｍｏｎｉａｅからの保護は用量応答性であった。これはこのモノクローナル抗体が、防御の原因であったことを示す（表３）。
【０１２２】
（表２．乳仔マウスにおける、抗３７ｋＤａモノクローナル抗体によって付与される、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｐｎｅｕｍｏｎｉａｅ血清型６Ｂに対する受動防御）
【０１２３】
【表２】

^ａチャレンジ用量（１．７×１０^３ｃｆｕ）または１０×菌血症用量１００％（ＢＤ_１００）。１群あたり５匹のマウスに、５０μｇの総抗体を与えた。すべてのＭＡｂは、ＩｇＧである。
【０１２４】
（表３．抗３７ｋＤａモノクローナル抗体８Ｇ１２Ｇ１１Ｂ１０の受動防御能の可能性に対する、第２用量の効果）
【０１２５】
【表３】

^ａ全ての乳仔マウスを、１０×ＢＤ_１００（２×１０^３ｃｆｕ）でチャレンジした。ＭＡｂを、チャレンジの２４時間前（前）およびチャレンジの２４時間後（後）に与えた。１０匹のマウス／群。
【０１２６】
（実施例１０．ファージディスプレイライブラリー）
ｐＩＩＩコーティングタンパク質（ＰａｒｍｌｙおよびＳｍｉｔｈ，１９８８）のＮ末端部分で位置する１５アミノ酸残基の挿入物を含むファージディスプレイライブラリーを、ベクターとしてファージＦＵＳＥ　５において構築した。このライブラリーを、３３ｂｐの合成ＢｇＩＩフラグメントをＦＵＳＥ　５に連結し、そしてＥ．ｃｏｌｉ　Ｋｑｌｌ／ｋａｎ＋細胞を、エレクトロポレーションによってトランスフェクトすることにより作製した。このファージ子孫は、ディスプレイライブラリーを含む。
【０１２７】
（実施例１１．バイオパニング（ｂｉｏｐａｎｎｉｎｇ）によるスクリーニング）
４サイクルのバイオパニングを、実施例１０のファージディスプレイライブラリーをＰｓａＡエピトープペプチドについてスクリーニングする目的で、ファージ使用される各ＭＡｂについて行った（ＳｍｉｔｈおよびＳｃｏｔｔ，１９９３、Ｍｅｔｈ．Ｅｎｚｙｍｏｌ．、２１７：２２８〜２５７）。ペトリ皿の基質を、実施例１において調製されるように、ストレプトアビジンおよび１０μｇのビオチン化抗ＰｓａＡ　ＭＡｂでコーティングした。残りのビオチン結合部位を、１．５ｍｌのＤ−ビオチン（１０ｍＭ）でブロックした。次いで、このファージライブラリー（１０^１１〜１０^１２個の形質転換単位）を、固定化したＭＡｂと共にインキュベートした。結合したファージを、ストレプトアビジンでコーティングされたプレートから、０．１Ｎ　ＨＣｌ、ｐＨ２．２で溶出した。この溶出されたファージを、力価測定および増幅し、次いで上記のように行ったさらに２回の選択に供した。使用されたビオチン化ＭＡｂの量は、第２回目および第３回目においてそれぞれ１ｎＭおよび１ｐＭであり、その結果、高親和性のペプチドのみが、最後のサイクルの終了時に結合された。
【０１２８】
（実施例１２．免疫原性ペプチドのアミノ酸配列）
実施例１１の手順を使用して得られた、ライブラリー由来の高親和性ペプチドを、増殖および配列決定した。それぞれのＭＡｂについて、選択プロセスから生じた１０個のファージ検体を、配列決定した。約１μｇの一本鎖ＤＮＡを、フェノール−クロロホルム抽出によって精製し、エタノール沈殿させ、そして７μｌの水に再懸濁した。全てのｆｄ−ｔｅｔ誘導ベクターに共通の、野生型ｐＩＩＩにおける領域から誘導されたＦＵＳＥ　５ベクター配列に相補的な２７マーのプライマーおよび^３５Ｓ　Ｓｅｑｕｅｎａｓｅ　バージョン２（Ｕ．Ｓ．Ｂｉｏｃｈｅｍｉｃａｌｓ、Ｃｌｅｖｅｌａｎｄ、ＯＨ）を使用して、配列決定反応を行った。得られた配列を、表４において示す。これらを、ＣｌｕｓｔａＩＶおよびｔＦａｓｔａプログラムを使用して、ＰｓａＡ株２および６Ｂの既知の配列と比較し、それぞれのペプチドが最も近く整列される、ＰｓａＡに関するエピトープを同定した。これらのエピトープ位置をまた、表４において示す。さらなる残基がこのタンパク質に存在し、ここでこのペプチド（配列番号７および配列番号８）の残基１３の後にギャップが出現する場合、ＭＡｂ（８Ｇ１２、６Ｆ６および１Ｅ７）を使用して得られるペプチドは、ＰｓａＡに最も整列する。
【０１２９】
（表４．ＭＡｂを用いたバイオパニングによって得られるペプチド配列）
【０１３０】
【表４】

（実施例１３．ＰｓａＡの免疫原性ペプチドでのマウスの免疫）
配列番号５、６、７、および８において示される配列を有するペプチドを、従来の方法によって（例えば、自動化ペプチド合成器を使用して）合成し得る。このペプチドを、逆相ＨＰＬＣによって精製し得、そして主要なピークを収集し得る。ペプチド配列を、自動化配列決定装置（例えば、Ｂｅｃｋｍａｎ　Ｉｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．，Ｍｏｕｎｔａｉｎ　Ｖｉｅｗ、ＣＡによって製造される自動化配列決定装置）を使用して、自動化ペプチド配列決定によって確認し得る。各ペプチドを、水溶性カルボジイミド試薬によって媒介されるカップリングを使用して、キーホールリンペットヘモシアニンのようなアジュバントに結合し得る。生じた結合体を、リン酸緩衝化生理食塩水　ｐＨ７．２に、最終濃度が約１８０μｇ／ｍｌとなるように溶解し得、そしてエマルジョン中に、約１：１の比でフロイント不完全アジュバント（Ｓｉｇｍａ　Ｃｈｅｍｉｃａｌ　Ｃｏ．，Ｓｔ　Ｌｏｕｉｓ，ＭＯ）と合わせ得る。ＢＡＬＢ／ｃマウスを、この懸濁液を用いて腹腔内に最初に免疫化し得、そして一月後、このマウスを、アジュバントを有さない約１１０μｇ／ｍｌの結合体を用い、ブーストし得る。
【０１３１】
（実施例１４．ファージディスプレイによって同定されたコンセンサスペプチドでのマウスの免疫）
ファージディスプレイによって同定されたいくつかのペプチドの配列由来のコンセンサス配列（配列番号８）を有するペプチドを、ＰｓａＡに対する免疫学的な応答を惹起するその能力について、試験した。上記の実施例１２に記載されるファージディスプレイ実験においてモノクローナル抗体８Ｇ１２、６Ｆ６および１Ｅ７に結合するペプチドに由来するこのコンセンサスペプチド（Ｌ　Ｖ　Ｒ　Ｒ　Ｆ　Ｖ　Ｈ　Ｒ　Ｒ　Ｐ　Ｈ　Ｖ　Ｅ　Ｓ　Ｑ；配列番号７）を、そのアミノ末端にＣＹＧＧスペーサーおよびラウロイル基を有するように合成した（Ｂｉｏ−Ｓｙｎｔｈｅｓｉｓ、Ｌｅｗｉｓｖｉｌｌｅ、ＴＸ）。このラウロイル基は、プロテオソームに対するペプチド基の疎水性複合体形成を増強する（Ｌｏｗｅｌｌ，Ｇ．Ｈ．ら、「Ｐｒｏｔｅｏｓｏｍｅ，ｈｙｄｒｏｐｈｏｂｉｃ　ａｎｃｈｏｒｓ，ｉｓｃｏｍｓ，ａｎｄ　ｌｉｐｏｓｏｍｅｓ　ｆｏｒ　ｉｍｐｒｏｖｅｄ　ｐｒｅｓｅｎｔａｔｉｏｎ　ｏｆ　ｐｅｐｔｉｄｅ　ａｎｄ　ｐｒｏｔｅｉｎ　ｖａｃｃｉｎｅｓ」、Ｎｅｗ　Ｇｅｎｅｒａｔｉｏｎ　Ｖａｃｃｉｎｅｓ，Ｗｏｏｄｒｏｗ，Ｇ．Ｍ．、Ｌｅｖｉｎｅ，Ｍ．Ｍ．（編）、Ｍａｒｃｅｌ　Ｄｅｋｋｅｒ，Ｉｎｃ．，Ｎｅｗ　Ｙｏｒｋ、１４１〜１６０頁（１９９０）；Ｌｏｗｅｌｌ，Ｇ．Ｈ．ら、「Ｐｅｐｔｉｄｅｓ　ｂｏｕｎｄ　ｔｏ　ｐｒｏｔｅｏｓｏｍｅｓ　ｖｉａ　ｈｙｄｒｏｐｈｏｂｉｃ　ｆｅｅｔ　ｂｅｃｏｍｅ　ｈｉｇｈｌｙ　ｉｍｍｕｎｏｇｅｎｉｃ　ｗｉｔｈｏｕｔ　ａｄｊｕｖａｎｔｓ」Ｊ．Ｅｘｐ．Ｍｅｄ．１６７：６５８（１９８８）；およびＺｏｌｌｉｎｇｅｒ，Ｗ．Ｄ．ら、「Ｃｏｍｐｌｅｘ　ｏｆ　ｍｅｎｉｎｇｏｃｏｃｃａｌ　ｇｒｏｕｐ　Ｂ　ｐｏｌｙｓａｃｃｈａｒｉｄｅ　ａｎｄ　ｔｙｐｅ　２　ｏｕｔｅｒ　ｍｅｍｂｒａｎｅ　ｐｒｏｔｅｉｎ　ｉｍｍｕｎｏｇｅｎｓ　ｉｎ　ｍａｎ」Ｊ．Ｃｌｉｎ．Ｉｎｖｅｓｔ．６３：８３６（１９７９））。一方、このシステイン基は、ペプチドの免疫原性を増強する（Ｌｏｗｅｌｌ，Ｇ．Ｈ．ら（１９９０））。Ｚｏｌｌｉｎｇｅｒ（Ｚｏｌｌｉｎｇｅｒら（１９９０））によって記載されるように、プロテオソームを、群Ｂ．ｍｅｎｉｎｇｏｃｏｃｃｉ、株９９Ｍ由来の外膜複合体小胞から調製した。合成的リポペプチドを、界面活性剤の存在下で、成分を合わせることによって、１：１（ｗ／ｗ）の比で、プロテオソームに対して複合体形成させた。この界面活性剤を、大規模な透析によって除いた（Ｌｏｗｅｌｌ，Ｇ．Ｈ．ら（１９８８））。
【０１３２】
３〜４週齢のＢＡＬＢ／ｃマウス（群あたりｎ＝４）を、１０、２５、または５０μｇのペプチド（配列番号５）／プロテオソーム複合体で、０週目にプライムした。次いで、それぞれの群におけるマウスを、１０、２５、または５０μｇのペプチド（配列番号５）／プロテオソーム複合体で、２週目および３週目にブーストした。０週目に２０μｇのＰｓａＡタンパク質でプライムしたマウスは、本研究のための陽性コントロールであった。陰性コントロールは、０週目に１０、２５、または５０μｇのプロテオソームでプライムし、そして２週目および３週目に１０、２５、または５０μｇのプロテオソームでブーストしたマウスから構成された。血清を、４週間にわたって毎週収集し、そして抗ＰｓａＡ特異的抗体を、酵素結合免疫吸着アッセイ（ＥＬＩＳＡ）によって試験した。
【０１３３】
抗ＰｓａＡ特異的ＥＬＩＳＡを、以下のように実行した：Ｎｕｎｃ　ｉｍｍｕｎｏ　Ｍａｘｉ　Ｓｏｒｂプレートを、５μｇ／ｍｌの精製されたネイティブなＰｓａＡタンパク質で、４℃で一晩コーティングした。プレートをＰＢＳ／Ｔｗｅｅｎ緩衝液（０．０１％のＴｗｅｅｎ−２０を含むＰＢＳ）で洗浄し、そして１％のＢＳＡを含むＰＢＳ／Ｔｗｅｅｎ緩衝液でブロッキングした。ＰＢＳ／Ｔｗｅｅｎ／ＢＳＡにおいて１：１０で始めたマウス血清の段階希釈物を、３７℃で１時間インキュベートした。このプレートを、ＰＢＳ／Ｔｗｅｅｎで４回洗浄した。ＰＢＳ／Ｔｗｅｅｎ／ＢＳＡ中に４０００：１で希釈した、西洋ワサビペルオキシダーゼに結合し、抗マウスＩｇＧおよびＩｇＭ（Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を、プレートに添加した。抗ＰｓａＡ抗体を、ｏ−フェニレンジアミン基質を用いて３０分間、暗所で検出した。吸光度を、Ｍｉｃｒｏｐｌａｔｅ　Ｅ１３１１（Ｂｉｏｔｅｋ，Ｗｉｎｏｏｓｋｉ，ＶＴ）で、４９０ｎｍで読み取った。
【０１３４】
ＭＡｂ　８Ｇ１２、１Ｅ７および６Ｆ６によって選択されたペプチドの全ての配列は、ＰｓａＡ分子の同じ領域に整列した。ＭＡｂ　８Ｇ１２によって選択された１つのペプチドは、上に示されるように、１Ｅ７および６Ｆ６によって選択されるペプチド（配列番号８）と同じ配列を有した。これらのペプチドとＰｓａＡとの間の相同性を決定し、これは６アミノ酸に存在した。この領域を使用して、コンセンサスペプチド配列（配列番号８）を規定した。このペプチドを、Ｎ末端ラウロイル基およびＣＹＧＧモチーフを用いて合成し、プロテオソームに複合体形成させ、そして免疫原性を改良した。マウスを、１０、２５、または５０μｇの、プロテオソームに複合体形成しているコンセンサスペプチドで免疫した。ペプチド免疫マウス由来の血清は、抗ＰｓａＡ応答を示した。本研究の結果を、表５および６において示す。このペプチド免疫マウスは、ＩｇＧ応答を発達させ、力価は４週目に１５９から２５２の範囲に及んだ。この力価を、プロテオソームバックグラウンド値に関してデータを標準化した後で計算した。３つの用量の群のうち、５０μｇのペプチド用量群に対する免疫応答が、最も高かった。しかし、このペプチドに対する抗ＰｓａＡ応答は、ＰｓａＡタンパク質に対する免疫応答よりも有意に低かった（ｐ＜０．０５）。
【０１３５】
（表５．ＰｓａＡに対するＩｇＭの力価）
【０１３６】
【表５】

このデータを、２０μｇのＰｓａＡで免疫したマウス由来のコントロール血清の２５％の力価として示す。
【０１３７】
（表６．ＰｓａＡに対するＩｇＧの力価）
【０１３８】
【表６】

表５におけるように、このデータを、２０μｇのＰｓａＡで免疫したマウス由来のコントロール血清の２５％の力価として示す。
【０１３９】
（実施例１５．ファージディスプレイによって同定されたペプチドの脂溶化した形態および脂溶化していない形態での、マウスの免疫（Ｓｒｉｖａｓｔａｖａ，Ｎ．ら、Ｈｙｂｒｉｄｏｍａ　１９：２３、２０００からのデータ）
配列番号５（Ｔ　Ｖ　Ｓ　Ｒ　Ｖ　Ｐ　Ｗ　Ｔ　Ａ　Ｗ　Ａ　Ｆ　Ｈ　Ｇ　Ｙ）のアミノ酸配列を有し、そしてＭＡｂ　４Ｅ９を使用したファージディスプレイによって選択されたペプチドを、Ｓ．ｐｎｅｕｍｏｎｉａｅチャレンジからマウスを防御するその能力について試験した。このペプチドの２つの形態を合成した：一方は、そのＮ末端にパルミトイル残基を有し（「配列番号５−脂溶化（ｌｉｐｉｄａｔｅｄ）」）、そしてもう一方は、このような誘導体化を有さない（「配列番号５−未脂溶化（ｕｎｌｉｐｉｄａｔｅｄ）」）。これらのペプチドを、Ｓｔｅｗａｒｔら、「Ｓｏｌｉｄ　ｐｅｐｔｉｄｅ　ｓｙｎｔｈｅｓｉｓ」第２版、Ｐｉｅｒｃｅ　Ｃｈｅｍｉｃａｌ　Ｃｏ．，Ｒｏｃｋｆｏｒｄ，ＩＬ（１９８４）に記載されるように固相Ｆ−ｍｏｃ化学を使用し、ＡＣＴモデル３９６ＨＰＳペプチド合成器（Ａｄｖａｎｃｅｄ　ＣｈｅｍＴｅｃｈ，Ｌｏｕｉｓｖｉｌｌｅ，ＫＹ）を使用して合成した。モノパルミチン酸を含む上記に記載されるペプチドの脂溶化バージョンを、パルミチン酸（Ｓｉｇｍａ　Ｃｈｅｍｉｃａｌｓ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を、上記に記載される標準的なアミノ酸結合（Ｖｅｒｈａｕｌら（１９９５））に関してと同じ反応条件を使用して樹脂結合ペプチドの脱保護されたアミノ末端に結合することによって、合成した。配列を、Ｐｏｒｔｏｎモデル＃２０９０自動化ペプチドシーケンサー（Ｂｅｃｋｍａｎ　Ｉｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．，Ｍｏｕｎｔａｉｎ　Ｖｉｅｗ，ＣＡ）を使用する、自動化されたペプチド配列決定によって検証した。ペプチドを、リン酸緩衝化生理食塩水　ｐＨ７．２（ＰＢＳ）中に、最初の用量について１．０ｍｇ／ｍｌ、そして続く用量について０．５ｍｇ／ｍｌの最終濃度になるように懸濁した。
【０１４０】
ペプチド配列番号５−脂溶化および配列番号５−未脂溶化の、Ｓ．ｐｎｅｕｍｏｎｉａｅチャレンジに対して防御する能力を分析するために、１０週齢のＮＤ−４マウス（Ｓｗｉｓｓ　Ｗｅｂｓｔｅｒ）を、３用量レジメンを使用して免疫した。試験マウス（各ペプチドについてｎ＝１５）は、０日目に１００μｇのペプチドの初回用量を受け、続いて３週目および５週目で、５０μｇのペプチドのブースター用量を受けた。このペプチド配列番号５−脂溶化を、１００μｌ　ＰＢＳ　０．０１Ｍ、ｐＨ７．２に懸濁し、一方、未脂溶化ペプチド（配列番号５−未脂溶化）を、アジュバント　アルヒドロゲル（ａｌｈｙｄｒｏｇｅｌ）（２％；＃Ａ１０９０ＢＳ、Ａｃｃｕｒａｔｅ　Ｃｈｅｍｉｃａｌ　ａｎｄ　Ｓｃｉｅｎｔｉｆｉｃ　Ｃｏｍｐａｎｙ，Ｗｅｓｔｂｕｒｙ，ＮＹ）と共に、ＰＢＳ中に６．３ｍｇ／ｍｌの濃度で混合し、このペプチドの免疫原性を増強させた。コントロールマウス（ｎ＝１２）を、ペプチドを用いないこと以外は同様に免疫化した。各マウスを、肩の間に皮下で免疫した。最終のブーストの１週間後、全てのマウスを、４．９×１０^６ｃｆｕのＳ．ｐｎｅｕｍｏｎｉａｅの株ＰＬＮ−Ｄ３９（Ｊａｍｅｓ　Ｐａｔｏｎ、Ｗｏｍｅｎ’ｓ　ａｎｄ　Ｃｈｉｌｄｒｅｎ’ｓ　Ｈｏｓｐｉｔａｌ，Ｎｏｒｔｈ　Ａｄｅｌａｉｄｅ，Ｓ．Ａ．Ａｕｓｔｒａｌｉａによって寄贈された）（これはＤ３９のニューモライシン（ｐｎｅｕｍｏｌｙｓｉｎ）陰性誘導体である）でチャレンジした。この５日後に安楽死させ、そして鼻洗浄物（ｎａｓａｌ　ｗａｓｈ）の培養をした。ＰＢＳ鼻洗浄を、Ｗｕ，Ｈ．Ｙ．ら（「Ｅｓｔａｂｌｉｓｈｍｅｎｔ　ｏｆ　ａ　Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｐｎｅｕｍｏｎｉａｅ　ｎａｓｏｐｈａｒｙｎｇｅａｌ　ｃｏｌｏｎｉｚａｔｉｏｎ　ｍｏｄｅｌ　ｉｎ　ａｄｕｌｔ　ｍｉｃｅ」、Ｍｉｃｒｏｂ．Ｐａｔｈｏｇ．２３：１２７（１９９７））の方法によって行った。各洗浄物を、１：４８６の最終希釈物なるように３倍希釈した。５０μｌの各希釈物を、血液寒天＋ゲンタマイシンプレート（５％の脱線維したヒツジ血液および０．５％のゲンタマイシンを補充したトリプチカーゼ（Ｔｒｙｐｔｉｃａｓｅ）ダイズ寒天）上で培養した。免疫したマウスおよびプラセボ（ＰＢＳ免疫化コントロール）におけるＮＰコロニー形成および保因（ｃａｒｒｉａｇｅ）からのデータを、ｔ−検定またはＭａｎｎ−Ｗｈｉｔｎｅｙ順位和検定のいずれかを使用して分析した。鼻咽頭の保因は、鼻あたりのコロニー形成単位の数である。鼻咽頭のコロニー形成は、２５μｌの鼻洗浄物において少なくとも１ｃｆｕを形成するか否かに依存して、マウスについて陽性または陰性のいずれかである。
【０１４１】
ペプチド配列番号５−脂溶化および配列番号５−未脂溶化の、Ｓ．ｐｎｅｕｍｏｎｉａｅチャレンジに対して防御する能力を分析するために、Ｓｗｉｓｓ　Ｗｅｂｓｔｅｒマウスを、配列番号５−脂溶化および配列番号５−未脂溶化、およびＰｓａＡで、上記のように免疫化した。ペプチド配列番号５−脂溶化で免疫されたマウスは、プラセボコントロールと比較した場合、細菌の鼻保因において統計学的に有意な減少（ｐ＜０．０５）を示した（表７）。さらに、ペプチド配列番号５−未脂溶化で免疫したマウスは、Ｓ．ｐｎｅｕｍｏｎｉａｅ血清型２によるコロニー形成に耐性である機会が４０％高かった（表７）。これは、プラセボコントロールと比較した場合、統計学的に有意であった（ｐ＜０．０５、Ｆｉｓｈｅｒの直接確立検定、両側検定）。肺炎球菌の鼻コロニー形成の減少は、非脂溶化ペプチド配列番号５−未脂溶化で免疫したマウスにおいて見出された；しかし、この減少は、プラセボコントロールと比較した場合、統計学的に有意ではなかった（ｐ＝０．４８）（表７）。
【０１４２】
（表７．Ｓｗｉｓｓ−Ｗｅｂｓｔｅｒマウスにおける防御研究の要約^＊）
【０１４３】
【表７】

^＊このデータは、３回の実験の保因（ｃｆｕ／鼻）の相乗平均として示される。コロニー形成を、１ｃｆｕ／２５μｌ鼻洗浄物として規定する。この有意性を、Ｍａｎｎ−Ｗｈｉｔｎｅｙ　Ｕ順位和検定を使用して計算する（ｐ＜０．０５）。
^＊＊Ｎ末端の付加的なシステイン残基でのモノパルミチン酸を用いた脂溶化。この脂溶化されたペプチドは、システイン（Ｃ）残基がこのペプチドのアミノ末端にあるように、示された配列表からのアミノ酸配列のアミノ末端に結合したアミノ酸配列ＣＳＳを含んでいた。
【０１４４】
（実施例１６　複数抗原性ＰｓａＡペプチドでのマウスの免疫化）
配列番号５、配列番号６、および配列番号７に示された配列を有するペプチドの、Ｓ．ｐｎｅｕｍｏｎｉａｅの攻撃からマウスを防御する能力を、脂溶化形態もしくは複数抗原性ペプチド（ＭＡＰ）形態で別個に投与するか、またはＭＡＰと結合して投与した場合に、調べた。配列番号５、配列番号６および配列番号７で与えらる配列を有するペプチドを、実施例１５に記載されるような、類似の試薬、装置、手順を使用して合成した。２つのアーム、３つアーム、または４つアームのうちいずれかを含む複数抗原性ペプチド（ＭＡＰ）（図１Ａ〜１Ｃ）を、Ｔａｍ，Ｊ．Ｐ．、「Ｍｕｌｔｉｐｌｅ　ａｎｔｉｇｅｎｉｃ　ｐｅｐｔｉｄｅ　ｓｙｓｔｅｍ：Ａ　ｎｏｖｅｌ　ｄｅｓｉｇｎ　ｆｏｒ　ｓｙｎｔｈｅｔｉｃ　ｐｅｐｔｉｄｅ　ｖａｃｃｉｎｅｓ　ａｎｄ　ｉｍｍｕｎｏａｓｓａｙ」　Ｓｙｎｔｈｅｔｉｃ　Ｐｅｐｔｉｄｅｓ　Ａｐｐｒｏａｃｈｅｓ　ｔｏ　Ｂｉｏｌｏｇｉｃａｌ　Ｐｒｏｂｌｅｍｓ，Ｊ．Ｐ．ＴａｍおよびＥ．Ｔ．Ｋａｉｓｅｒ編，Ａｌａｎ　Ｒ．Ｌｉｓｓ，Ｉｎｃ．，Ｎｅｗ　Ｙｏｒｋ，３−１８（１９８９）の方法に従って合成した。この実施例に記載される全てのＭＡＰは、合成を容易にするためのさらなるノルロイシン（ＮＬｅ）残基をカルボキシ末端に含む（図ｌＡ〜図１Ｃ）。ミョウバンを、非脂溶化ペプチドのためにこの懸濁物に添加した。
【０１４５】
別個の群のＮＤ−４マウス（ペプチド群およびコントロール群当たりｎ＝８）を、第０週、第３週、第５週の３回、それぞれ１００μｇ、５０μｇ、または５０μｇの非脂溶化ペプチドまたは脂溶化ペプチドのいずれかで、皮下にて免疫化した。第３の用量の１週間後、マウスを、１０μｌの０．８５％生理食塩水中に懸濁した、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｐｎｅｕｍｏｎｉａｅの血清型２、血清型４、または血清型６Ｂの１０^６ｃｆｕで鼻腔内（ＩＮ）にて攻撃した。この５日後に、安楽死させ、そして鼻腔内洗浄液を培養した。鼻咽頭（ＮＰ）のコロニー形成およびキャリッジを、実施例１５に記載されるように、分析した。
【０１４６】
非脂溶化または脂溶化した、単相性ペプチドのＭＡＰ（すなわち、均質）および二相性ペプチドＭＡＰ（すなわち、２つの異なるペプチド配列を含む不均質ＭＡＰ）を用いた免疫化は、鼻腔内キャリッジに対するインビボ保護（すなわち、ｃｆｕの減少）によって確証が得られるような、Ｓ．ｐｎｅｕｍｏｎｉａｅの攻撃に対する免疫学的応答を生じる。最も強い免疫学的応答を、二相性ペプチドＭＡＰの免疫化で観察した。ＮＰコロニー形成の減少を、脂溶化ペプチドおよび非脂溶化ペプチド、ならびに様々なＭＡＰ構築物で、観察し、そしてこの減少は、これらの間で変化した。非脂溶化単相性ペプチド（すなわち、均質な）ＭＡＰで免疫化することによって、ＮＰコロニー形成が４０％と９３％との間に減少した（表８）。脂溶化単相性ペプチドで免疫化することによって、ＮＰコロニー形成が、４８％と９４％との間に減少した。キャリッジで最も大きい減少は、二相性ペプチドＭＡＰで観察された。非脂溶化二相性ペプチドＭＡＰの免疫化によって、ＮＰコロニー形成が、６８％と９１％との間に減少した（表９）。二相性ペプチドＭＡＰの免疫化で観察された、このＮＰコロニー形成の減少は、試験した全ての血清型および二相性ペプチドについて統計的に有意（ｐ＜０．０５）であった。
【０１４７】
ＰｓａＡに対するモノクローナルを使用するファージディスプレイ分析によって同定される、実験で使用したＰｓａＡペプチドは、免疫原性であり、そしてこれは、試験したＳ．ｐｎｅｕｍｏｎｉａｅの３つの血清型のキャリッジを減少した。さらに、二相性ペプチドＭＡＰは、単相性ペプチドＭＡＰよりも、キャリッジの減少においてより効率的であった。
【０１４８】
（表８．鼻咽頭（ＮＰ）コロニー形成の減少（％）：コントロールに比較した非脂溶化４アーム均質ＭＡＰ（Ｐ値））
【０１４９】
【表８】

^＊非脂溶化ペプチド（ＮＬ）。
^＊＊コントロールからの統計的有意差（Ｐ≦０．０５）を、ｔ検定またはＭａｎｎ−Ｗｈｉｔｎｅｙ順位和検定で算出した。
^＊＊＊配列番号５の均質４アームＭＡＰ。
^＊＊＊＊配列番号６の均質４アームＭＡＰ。
^{＊＊＊＊＊}配列番号７の均質４アームＭＡＰ。
【０１５０】
（表９．コントロールに対して比較した本発明の脂溶化ペプチドＭＡＰおよび二相性ペプチドＭＡＰのＮＰコロニー形成の減少（％）^＊）
【０１５１】
【表９】

^＊（Ｐ値）。
^＊＊Ｎ末端のさらなるシステイン残基において、モノパルミチン酸で脂溶化。この脂溶化ペプチドは、このシステイン（Ｃ）残基がこのペプチドのアミノ末端になるように、示された配列表由来のアミノ酸配列のアミノ末端に結合するアミノ酸配列ＣＳＳを含む。
^＊＊＊図１Ａに示されるような、配列番号５および配列番号６の不均質４アームＭＡＰ。
^＊＊＊＊図１Ｂに示されるような、配列番号５および配列番号９の不均質４アームＭＡＰ。
【０１５２】
（実施例１７．三相性ペプチドＭＡＰでのマウスの免疫化）
均質（単相性ペプチド）３アームＭＡＰおよび不均質（二相性ペプチドまたは三相性ペプチド）３アームＭＡＰをまた、Ｓ．ｐｎｅｕｍｏｎｉａｅの攻撃に対する防御を提供するために使用し得る。例えば、単相性ペプチドＭＡＰおよび二相性ペプチドＭＡＰについて上で記載したように、配列番号５〜配列番号１０のうちの任意の組み合わせを含む三相性ペプチド３アームＭＡＰまたはそれらの免疫原性フラグメント（例えば、各々が配列番号５に示されるアミノ酸配列を有する、３アームを含む均質三相性ペプチドＭＡＰ、または配列番号５の配列を有する第１のアーム、配列番号９の配列を有する第２のアーム、および配列番号１０の配列を有する第３のアームを含む不均質三相性ペプチドＭＡＰ；例えば、図１Ｃ参照のこと）を、実施例１５および実施例１６に記載されるような、類似の試薬、装置、および手順を用いて、合成し得、そして試験し得る。三相性ペプチドの組み合わせの他のこのような例は、以下であるが、これらに限定されない：配列番号５を含む第１のアーム、配列番号６を含む第２のアーム、および配列番号７、配列番号８、または配列番号９を含む第３のアーム；配列番号５を含む第１のアーム、配列番号１０を含む第２のアーム、および配列番号７、配列番号８、または配列番号９を含む第３のアーム；配列番号６を含む第１のアーム、配列番号７を含む第２のアーム、および配列番号９または配列番号１０を含む第３のアームなど。
【０１５３】
（実施例１８．ＰｓａＡ由来のペプチドを用いた皮下免疫化による、マウスにおける肺炎球菌キャリッジの阻害）
本発明者らは、免疫化したマウスにおける、肺炎球菌（Ｐｎｃ）鼻咽頭（ＮＰ）キャリッジを阻害するＭＡＰの能力について、脂溶化形態または脂溶化形態の単独、または互いに組み合わせ（２つの二相性ペプチドおよび三相性ペプチド）のいずれかである、３つのＭＡＰを試験した。ＮＰキャリッジの阻害は、ＭＡＰの組み合わせおよび二相性ペプチドを使用して、均質ＭＡＰ単独に比べ、劇的に増大された。これらの観察は、この抗ＰｓａＡ　ＭＡｂファージディスプレイ発現ペプチドは、免疫原性であり、そしてマウスにおけるＮＰキャリッジを有意に減少することを示した。これらのＰｓａＡペプチドは、Ｐｎｃキャリッジ、中耳炎、および／また侵襲性Ｐｎｃ疾患の減少および／または除去について、ヒトおよび／または他の動物にとって、おそらくはワクチンとして有用である。
【０１５４】
（Ａ）材料および方法）
（細菌性単離体および増殖条件）
Ｓ．ｐｎｅｕｍｏｎｉａｅの３つの単離体（血清型２、血清型４および血清型６Ｂを表す）を、ＮＰキャリッジ実験のために使用した。血清型２である単離体ＰＬＮ−Ｄ３９は、Ｊａｍｅｓ　Ｐａｔｏｎ（Ｗｏｍｅｎ’ｓ　ａｎｄ　Ｃｈｉｌｄｒｅｎ’ｓ　Ｈｏｓｐｉｔａｌ，Ｎｏｒｔｈ　Ａｄｅｌａｉｄｅ，Ｓ．Ａ．，Ａｕｓｔｒａｌｉａ）の厚意によって提供され；そして、血清型４である単離体ＤＳ２３４１−９４、および６Ｂである単離体ＤＳ　１７５６−９４は、Ｒｉｃｈａｒｄ　Ｆａｃｋｌａｍ（Ｃｅｎｔｅｒｓ　ｆｏｒ　Ｄｉｓｅａｓｅ　Ｃｏｎｔｒｏｌ　ａｎｄ　Ｐｒｅｖｅｎｔｉｏｎ，Ａｔｌａｎｔａ，ＧＡ）の厚意によって提供された。これらの選択された単離体は、代表的な株であり、発明者らの実験室および他の場所において動物モデル実験において頻繁に使用されていた。多数の実験が同じロットの細胞から開始され得たことを確実にするために、各肺炎球菌単離体の標準化ストック培養物を、上述したように調製した（Ｊｏｈｎｓｏｎ　ＳＥら、Ｊ．Ｉｎｆｅｃｔ．Ｄｉｓ．１８０：１３３−４０，１９９９）。実験を開始する際に、ストック細胞を、血液寒天培地（ＢＡ，５％脱フィブリンヒツジ血液を補充したＴｒｙｐｔｉｃａｓｅダイズ寒天；ＢＢＬ，Ｍｉｃｒｏｂｉｏｌｏｇｙ　Ｓｙｓｔｅｍｓ，Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ　ａｎｄ　Ｃｏ．，Ｃｏｃｋｅｙｓｖｉｌｌｅ，ＭＤ）上で培養し、１０％　Ｌｅｖｉｎｔｈａｌ基礎培地（ＢＨＩ−１０Ｌ；ＢＢＬ）を補充した脳心臓注入ブロス（ｂｒａｉｎ　ｈｅａｒｔ　ｉｎｆｕｓｉｏｎ　ｂｒｏｔｈ）（ＢＨＩ；ＢＢＬ）に移し、そして以前に記載したように動物接種のために操作した（Ｊｏｈｎｓｏｎ　ＳＥら、前出）。
【０１５５】
（動物）
成体Ｓｗｉｓｓ　Ｗｅｂｓｔｅｒマウス（ＮＤ−４；雌性；８週齢）を、Ｈａｒｌａｎ　Ｓｐｒａｇｕｅ　Ｄａｗｌｅｙ，Ｉｎｃ．，Ｉｎｄｉａｎａｐｏｌｉｓ，ＩＮから得た。到着時に、マウスを１ケージ８当たり匹の群に配置した。全ての動物を、標準的な条件（２５℃、相対湿度約４０％）下で、食料および水の任意の摂取を伴って収容した。全てのマウスが、実験前の１週間で、これらの新環境に対して順応することを可能にした。
【０１５６】
（ペプチド合成）
前述のペプチド（Ｐ１、Ｐ２、およびＰ３で示されるベースペプチド）のアミノ酸配列を、テンプレートとして使用する場合、脂溶化形態および非脂溶化形態の両方であって、３つの、４−アームで均質な複数抗原性ペプチド（ＭＡＰ）を、非脂溶化二相性（４−アーム不均質）ペプチド構築物および三相性ペプチド（３−アーム均質）構築物と共に合成し、そして、マウスＮＰキャリッジモデルで研究した。これらのＭＡＰを、以前に記載された（Ｂａｒａｎｙ，ＧおよびＭｅｒｒｉｆｉｅｌｄ，ＲＢ，Ｔｈｅ　Ｐｅｐｔｉｄｅｓ，第１巻，Ｇｒｏｓｓ，ＥおよびＭｅｉｅｎｈｏｆｅｒ，Ｊ，編，Ｎｅｗ　Ｙｏｒｋ：Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ，１９８０，ｌ−２８４頁；Ｖｅｒｈｅｕｌ，ＡＦら、Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈ．１８２：２１９−２２６，１９８５；ならびにＲｅｅｄ，ＲＣら、Ｖａｃｃｉｎｅ　１５：４８２−４８８，１９９７）のような、標準Ｆｍｏｃプロトコルおよび改変Ｆｍｏｃプロトコルを使用する、ＡＣＴ　３９６マルチプルペプチド合成機（Ａｄｖａｎｃｅｄ　ＣｈｅｍＴｅｃｈ，Ｌｏｕｉｓｖｉｌｌｅ，ＫＹ）で合成した。この二相性ペプチド構築物および三相性ペプチド構築物を、Ｔａｍの方法（Ｔａｍ　ＪＰ，Ｍｕｌｔｉｐｌｅ　ａｎｔｉｇｅｎｉｃ　ｐｅｐｔｉｄｅ　ｓｙｓｔｅｍ：Ａ　ｎｏｖｅｌ　ｄｅｓｉｇｎ　ｏｆ　ｓｙｎｔｈｅｔｉｃ　ｐｅｐｔｉｄｅ　ｖａｃｃｉｎｅｓ　ａｎｄ　ｉｍｍｕｎｏａｓｓａｙ，Ｔａｍ，ＪＰおよびＫａｉｓｅｒ，ＥＴ編、Ｎｅｗ　Ｙｏｒｋ：Ａｌａｎ　Ｒ．Ｌｉｓｓ，Ｉｎｃ．，１９８９，３−１８頁）を使用して、ピペリジンおよびヒドラジンでの、Ｆｍｏｃ−Ｌｙｓ（Ｄｄｅ）またはＦｍｏｃ−Ｌｙｓ（ｉｖＤｄｅ）（Ｎｏｖａｂｉｏｃｈｅｍ，Ｓａｎ　Ｄｉｅｇｏ，ＣＡ）の差次的脱保護（ｄｉｆｆｅｒｅｎｔｉａｌ　ｄｅｐｒｏｔｅｃｔｉｏｎ）によって合成した。この粗ペプチドを、マトリックス支援レーザー脱離／イオン化飛行時間型質量分析、その後の、アミノ酸分析、高速液体クロマトグラフィー（ＨＰＬＣ）、キャピラリー電気泳動および／またはペプチド配列決定のうちの１つ以上の手順によって、合成の忠実度について分析した。必要である場合、このペプチドを、アセトニトリル／水／トリフルオロ酢酸緩衝系を使用して、半分取ＨＰＬＣによって精製した。
【０１５７】
構造的に、このＭＡＰは、互いに対して相同であり、そしてＰ１、Ｐ２またはＰ３のいずれかからなる、４つの分岐を含む。脂溶化形態（Ｐ４３、Ｐ４４、およびＰ４５として示される）および非脂溶化形態（Ｐ４６、Ｐ４７、およびＰ４８として示される）は、前者が、各ベースペプチド単位のＮ末端において、３アミノ酸リンカー（システイン−セリン−セリン）によって結合された、一置換パルミトイル残基を有することを除いて構造上は同一である（図２Ａおよび図２Ｂ）。このＭＡＰを、パルミチン酸で脂溶化し、ペプチド免疫原性におけるアジュバントの効果およびＮＰキャリッジに対するその後の影響を研究した。Ｐ７９およびＰ８０と称される二相性ペプチドは、ベースペプチドの組み合わせ（Ｐ１およびＰ２、またはＰ１およびＰ３）である（図３）。Ｐ７９は、Ｐ１およびＰ２の各々の２単位を含み、一方Ｐ８０中では、Ｐ２が、Ｐ３で置換されている。三相性ペプチド（Ｐ８１として同定され、そして３つの分岐からなる）は、ベースペプチドの各々の１つの単位から構成されている（図４）。全ての場合において、このペプチドの分岐を、３−アームのロイシン−ノルロイシンコアまたは４−アームのロイシン−ノルロイシンコアのいずれかに基づいて合成した（図１〜図３）。
【０１５８】
（マウス免疫化および攻撃）
マウスＮＰキャリッジモデル（前述）を使用して、ＮＰ　Ｐｎｃキャリッジの阻害における、免疫原性ＰｓａＡペプチドの影響を決定した（Ｌｉｐｓｉｔｃｈ，Ｍら，Ｖａｃｃｉｎｅ　１８：２８９５−９０１，２０００）。マウスを、３用量レジメンに基づいて免疫化した。試験マウス（各ペプチドまたはペプチド構築物についてｎ＝８）は、第０日目での１００μｇの開始用量に続き、初回の用量から３週間後および５週間後に、適切なペプチドの５０μｇのブースター用量を受けた。この脂溶化ＭＡＰを、１００μＬの無菌０．０１Ｍ，ｐＨ　７．２，リン酸緩衝生理食塩水（ＰＢＳ）中に再懸濁したが、全ての他のペプチドを、１００μＬの、ＰＢＳ中で６．３ｍｇ／ｍＬのアジュバントミョウバン（アルヒドロゲル（ａｌｈｙｄｒｏｇｅｌ）−２％，＃Ａ１０９０Ｂ，Ａｃｃｕｒａｔｅ　Ｃｈｅｍｉｃａｌ　ａｎｄ　Ｓｃｉｅｎｔｉｆｉｃ　Ｃｏｍｐａｎｙ，Ｗｅｓｔｂｕｒｙ，ＮＹ）と混合し、このペプチドの免疫原性を増大させた。コントロールマウス（ｎ＝８）を、同様に免疫化したが、ペプチドは用いなかった。各マウスを、肩の間の皮下で免疫化した。最終ブースト後１週間目に、全てのマウスを、鼻腔内から、１０μＬの０．９％塩化ナトリウム緩衝液（ＳＣＢ；Ａｂｂｏｔｔ　Ｌａｂｏｒａｔｏｒｉｅｓ，Ｎｏｒｔｈ　Ｃｈｉｃａｇｏ，ＩＬ）に懸濁された適切なＳ．ｐｎｅｕｍｏｎｉａｅ単離体の１〜３×１０^６コロニー形成単位（ｃｆｕ）で攻撃した。攻撃接種物を、１０倍に連続希釈し、ＢＡプレート上にプレートし、攻撃用量を決定した。
【０１５９】
（鼻腔洗浄および培養）
攻撃後５日目に、各マウスを安楽死させ、そしてＷｕら（Ｗｕ，ＨＹら、Ｍｉｃｒｏｂ．Ｐａｔｈｏｇ．２３：１２７−３７，１９９７）によって記載されたように１００μＬのＳＣＢでこの鼻咽頭腔を洗浄した。この洗浄物を、３倍に連続希釈し、最終的に１：４８６の希釈とした。５０μｌの各希釈物を、ＢＡ＋ゲンタマイシンプレート（０．５％ゲンタマイシンを補充したＢＡプレート）上で培養した。培養物を、５％ＣＯ_２雰囲気下かつ高湿度下にて、３７℃で一晩インキュベートした。可能な場合、クローンを、各希釈物のについて計数し、記録した。特定マウスのためのキャリッジを、各希釈物の計数を非希釈レベルに調整し、そして平均化した後に、５０μＬの回収した鼻腔洗浄物中のｃｆｕ数として規定した。キャリッジアッセイの検出感度は、４０ｃｆｕ／（ｍｌ鼻腔洗浄物）である。
【０１６０】
（統計方法）
Ｐｎｃキャリッジの阻害を、免疫化した試験マウスとこれらの対となる免疫原がないコントロールマウスとの間のＮＰキャリッジにおける有意差を算出することによって決定した。群の間の有意差は、Ｍａｎｎ−Ｗｈｉｔｎｅｙ順位和検定によって計算された。危険率をＰ＜０．０５に設定した。統計計算を、ＳｉｇｍａＳｔａｔソフトウエア，バーション２．０（Ｊａｎｄｅｌ　Ｓｃｉｅｎｔｉｆｉｃ　Ｃｏ．，Ｓａｎ　Ｒａｆａｅｌ，ＣＡ）を使用して実行した。分析は、Ｐｎｃ血清型および免疫原処方物の各々についての単一の試験から生成されたデータに基づく。
【０１６１】
（Ｂ）結果）
（ＮＰキャリッジ阻害におけるＭＡＰ効果）
予備的な実験において、ＭＡＰ　Ｐ４３に対する免疫学的応答を、マウスを上記のようなスケジュール（データは示さない）に従って免疫化した場合に、マウスにおいて観察した（力価は一貫して１：５１２００以上であった）。これらの結果に基づいて、Ｐｎｃキャリッジ阻害実験を、成体マウスにおいて記載のペプチドで開始した。マウスを非脂溶化ＭＡＰであるＰ４６、Ｐ４７、またはＰ４８で免疫化し、Ｓ．ｐｎｅｕｍｏｎｉａｅの血清型２、血清型４、または血清型６Ｂで攻撃した場合、有意な（Ｐ＜０．０５）なキャリッジ阻害を、コントロールと比較し、かつＮＰ腔でコロニー形成するｃｆｕのメジアン数で規定するときにキャリッジについて６０％を超える減少が存在した場合に、観察した（表１０）。Ｐ４６は、有意（Ｐ＜０．０５）に、血清型２および血清型４のＮＰコロニー形成を減少したが、血清型６Ｂのコロニー形成（Ｐ＝０．１１）は有意に減少しない。Ｐ４７およびＰ４８は、有意（Ｐ＜０．０５）に、各々１つの血清型、すなわち、それぞれ、血清型４および血清型６Ｂのコロニー形成を減少した。マウスを脂溶化ＭＡＰ　Ｐ４３、Ｐ４４、またＰ４５で免疫化した場合に、同様の結果が観察された（表１１）。Ｐｎｃコロニー形成が６８％減少した場合、鼻咽頭キャリッジ阻害は、有意（Ｐ＜０．０５）であった。血清型４のキャリッジを、これらのＭＡＰおよび血清型６Ｂ、血清型Ｐ４３、およびＰ４４の各々によって有意に（Ｐ＜０．０５）阻害した。しかし、これらのＭＡＰは、血清型２に対して有意な（Ｐ＜０．０５）なキャリッジ阻害を実証しなかったが、Ｐ４５で免疫化したマウスにおいて血清型２のＮＰコロニー形成に７０％減少が存在した（Ｐ＝０．２０）が、これらの有意（Ｐ＜０．０５）に阻害された血清型について、ＮＰ腔にコロニー形成するＰｎｃ細菌の数の平均は、非脂溶化および脂溶化ＭＡＰについて、それぞれ、５８％および７１％に減少された。
【０１６２】
（ＮＰキャリッジ阻害におけるＭＡＰの組み合わせまたはポリペプチドの効果）
このＭＡＰによるＮＰキャリッジ阻害についての有望な結果から、ＭＡＰの組み合わせおよびポリペプチド構築物を使用して、さらなる研究を進めた。マウスがＭＡＰの組み合わせ（Ｐ４３＋Ｐ４４またはＰ４３＋Ｐ４４＋Ｐ４５）または二相性ペプチド（Ｐ７９またはＰ８０）で免疫化される全ての場合にいおいて、ＮＰキャリッジの有意（Ｐ＜０．０５）な阻害を観察した（表１１）。ＮＰコロニー形成のレベルは、１つ（５５％）を除いて、全ての場合について有意（Ｐ＜０．０５）に少なくとも６７％減少した。しかし、マウスにおいてＮＰキャリッジを阻害する三相性ペプチドＰ８１の能力は、二相性ペプチドの能力ほど効果的ではなく、血清型２および血清型４において、それぞれ５３％および８９％の有意な（Ｐ＜０．０５）阻害を実証した（表１１）。血清型６Ｂに対する鼻咽頭キャリッジ阻害は、３９％（Ｐ＝０．１４）のみであった。二相性ペプチド阻害および三相性ペプチド阻害が有意（Ｐ＜０．０５）であった、これらの例について、平均値は、ＮＰ腔にコロニー形成するＰｎｃ細菌の数の平均は、それぞれ８０％および６０％減少した。
【０１６３】
（表１０．マウスモデルにおける、非脂溶化ＰｓａＡ　ＭＡＰによるＰｎｃ　ＮＰキャリッジの阻害）
【０１６４】
【表１０】

^ａデータは、鼻咽頭腔由来の１００μｌの洗浄物中で回収されたコロニー形成単位（ｃｆｕ）／マウス（ｎ＝８）のメジアン数を表す。マウスを、皮下にて１０^６ｃｆｕで攻撃した。
^ｂリン酸緩衝生理食塩水またはミョウバンのいずれかを注射されたコントロールマウスに対する有意差（Ｐ＜０．０５）。Ｍａｎｎ−Ｗｈｉｔｎｅｙ順位和検定を、群の間の有意差の算出のために使用。
（表１１．マウスモデルにおける、脂溶化ＰｓａＡ　ＭＡＰによるＰｎｃ　ＮＰキャリッジの阻害）
【０１６５】
【表１１】

^ａデータは、鼻咽頭腔由来の１００μｌの洗浄物中で回収されたコロニー形成単位（ｃｆｕ）／マウス（ｎ＝８）のメジアン数を表す。マウスを、皮下にて１０^６ｃｆｕで攻撃した。
^ｂリン酸緩衝生理食塩水またはミョウバンのいずれかを注射されたコントロールマウスに対する有意差（Ｐ＜０．０５）。Ｍａｎｎ−Ｗｈｉｔｎｅｙ順位和検定を、群の間の有意差の算出のために使用した。
（表１２．マウスモデルにおける、ＰｓａＡ　ＭＡＰの組み合わせ、二相性ペプチドまたは三相性ペプチドよる、Ｐｎｃ　ＮＰ　キャリッジの阻害）
【０１６６】
【表１２】

^ａデータは、鼻咽頭腔由来の１００μｌの洗浄物中で回収されたコロニー形成単位（ｃｆｕ）／マウス（ｎ＝８）のメジアン数を表す。マウスを、皮下にて１０^６ｃｆｕで攻撃した。
^ｂリン酸緩衝生理食塩水またはミョウバンのいずれかを注射されたコントロールマウスに対する有意差（Ｐ＜０．０５）。Ｍａｎｎ−Ｗｈｉｔｎｅｙ順位和検定を、群の間の有意差の算出のために使用した。
【図面の簡単な説明】
【図１】
図１Ａは、配列番号５または配列番号６のいずれかの配列を有する交互ペプチド（ａｌｔｅｒｎａｔｉｎｇ　ｐｅｐｔｉｄｅ）のアームを有する二相性ペプチド異種ＭＡＰを示す図である。
図１Ｂは、配列番号５または配列番号９のいずれかの配列を有する交互ペプチドのアームを有する二相性ペプチド異種ＭＡＰを示す図である。
図１Ｃは、配列番号５の配列を有する第１アーム、配列番号１０の配列を有する第２アーム、および配列番号９の配列を有する第３のアーム有する三相性ペプチド異種ＭＡＰを示す図である。各ペプチド中のカルボキシ末端リジン（すなわち、Ｋ）残基は、ＭＡＰの２つのアーム間で共有される。ＮＬｅは、ノルロイシンである。
【図２Ａ】
図２Ａおよび図２Ｂは、非脂質化ＭＡＰの基本構造（図２Ａ）および脂質化ＭＡＰの基本構造（図２Ｂ）を示す図である。ここで、「Ｂ」は、Ｐ１、Ｐ２またはＰ３のいずれかとして示す３つの１５アミノ酸ベースペプチドを表し；「Ｊ」は、パルミトイル（パルミチン酸）基を表し、これは、Ｕとして示す３アミノ酸鎖リンカー（−システイン−セリン−セリン−）を介してベースペプチド（Ｐ１、Ｐ２およびＰ３）のＮ末端に連結されている（Ｔａｍ　ＪＰ，Ｍｕｌｔｉｐｌｅ　ａｎｔｉｇｅｎｉｃ　ｐｅｐｔｉｄｅ　ｓｙｓｔｅｍ：Ａ　ｎｏｖｅｌ　ｄｅｓｉｇｎ　ｏｆ　ｓｙｎｔｈｅｔｉｃ　ｐｅｐｔｉｄｅ　ｖａｃｃｉｎｅｓ　ａｎｄ　ｉｍｍｕｎｏａｓｓａｙ，Ｔａｍ，ＪＰ　ａｎｄ　Ｋａｉｓｅｒ，ＥＴ編，Ｎｅｗ　Ｙｏｒｋ：Ａｌａｎ　Ｒ．Ｌｉｓｓ，Ｉｎｃ．，１９８９，３−１８頁）；Ｐ４３およびＰ４６、Ｂ＝Ｐ１（配列番号５）；Ｐ４４およびＰ４７、Ｂ＝Ｐ２（配列番号６）；ならびにＰ４５およびＰ４８、Ｂ＝Ｐ３（配列番号７）。
【図２Ｂ】
図２Ａおよび図２Ｂは、非脂質化ＭＡＰの基本構造（図２Ａ）および脂質化ＭＡＰの基本構造（図２Ｂ）を示す図である。ここで、「Ｂ」は、Ｐ１、Ｐ２またはＰ３のいずれかとして示す３つの１５アミノ酸ベースペプチドを表し；「Ｊ」は、パルミトイル（パルミチン酸）基を表し、これは、Ｕとして示す３アミノ酸鎖リンカー（−システイン−セリン−セリン−）を介してベースペプチド（Ｐ１、Ｐ２およびＰ３）のＮ末端に連結されている（Ｔａｍ　ＪＰ，Ｍｕｌｔｉｐｌｅ　ａｎｔｉｇｅｎｉｃ　ｐｅｐｔｉｄｅ　ｓｙｓｔｅｍ：Ａ　ｎｏｖｅｌ　ｄｅｓｉｇｎ　ｏｆ　ｓｙｎｔｈｅｔｉｃ　ｐｅｐｔｉｄｅ　ｖａｃｃｉｎｅｓ　ａｎｄ　ｉｍｍｕｎｏａｓｓａｙ，Ｔａｍ，ＪＰ　ａｎｄ　Ｋａｉｓｅｒ，ＥＴ編，Ｎｅｗ　Ｙｏｒｋ：Ａｌａｎ　Ｒ．Ｌｉｓｓ，Ｉｎｃ．，１９８９，３−１８頁）；Ｐ４３およびＰ４６、Ｂ＝Ｐ１（配列番号５）；Ｐ４４およびＰ４７、Ｂ＝Ｐ２（配列番号６）；ならびにＰ４５およびＰ４８、Ｂ＝Ｐ３（配列番号７）。
【図３】
図３は、二相性ペプチドＰ７９およびＰ８０の基本構造を示す図である。ここで、ベースペプチドは、２アミノ酸リンカーのリジン−ノルロイシン（Ｋ−ＮＬｅ）によって一緒に連結されて、４分岐の二相性ペプチドを形成する（Ｓｒｉｖａｓｔａｖａ　Ｎら、Ｈｙｂｒｉｄｏｍａ　１９：２３−３１，２０００；ならびにＴａｍ　ＪＰ（前出））。Ｐ７９は、ベースペプチドＰ１およびＰ２の各々の２単位からなる。二相性Ｐ８０において、Ｐ２単位は、Ｐ３単位で置換される。
【図４】
図４は、三相性ペプチドＰ８１の基本構造を示す図である。ここで、ベースペプチドは、２アミノ酸リンカーのリジン−ノルロイシン（Ｋ−ＮＬｅ）によって一緒に連結されて、ベースペプチドＰ１、Ｐ２、およびＰ３からなる３分岐の三相性ペプチドを形成する（Ｓｒｉｖａｓｔａｖａ　Ｎ（前出）およびＴａｍ，ＪＰ（前出））。[0001]
(Field of Invention)
The present invention relates to multiple antigenic peptides (multi-antigenic peptides) (MAP) that can be administered as a vaccine conferring immunity against Streptococcus pneumoniae.
[0002]
(Background of the Invention)
Pneumococcal disease continues to be a leading cause of illness and death in the United States and around the world. Currently used polysaccharide vaccines have limited efficacy in children younger than 2 years of age and exhibit serotype-specific variation in efficacy between vaccinated individuals. For these reasons, alternative vaccine formulations have been sought that do not require the use of multiple capsular polysaccharides. One potential strategy involves the use of immunogenic species-common proteins as vaccine candidates. These proteins can be used in combination with other immunogenic proteins or as protein carriers in protein, polysaccharide, or oligosaccharide conjugate vaccines. Efficient vaccines that contain a common protein would require a formulation based on multiple capsular polysaccharides (as in current 23-valent polysaccharide vaccines) by providing broader protection against multiple serotypes Sex can be excluded. Furthermore, protein-based vaccines are T cell dependent and provide a memory response, thereby making vaccines more effective.
[0003]
Immunogenic species-common proteins have been identified from Streptococcus pneumoniae (Russell et al., 1990, “Monoclonal antigenic recognition a specifics-specific protein 21. Michio USA. Patent No. 5,422,427). 37 kDa S.P. The pneumoniae protein has been the focus of several studies. At present, it is named as pneumococcal surface adhesion protein A (PsaA). (This 37 kDa protein was referred to in US Pat. No. 5,422,427 as the pneumococcal feminine protein; this term is used interchangeably herein). Immunoblot analysis studies using anti-PsaA monoclonal antibodies have shown that PsaA is common to all 23 pneumococcal vaccine serotypes (Russell et al., 1990). Enzyme-linked immunosorbent assay studies have shown that patients with pneumococcal disease show an increase in convalescent serum antibody to PsaA compared to acute serum antibody levels (Tharpe et al., 1995, “ Purification and seroreactivity of pneumococcal surface adhesinA (PsaA) "Clin.Diagn.Lab.Immunol.3:. 227~229; and Tharpe et al., 1994," The utility of a recombinant protein in an enzyme immunoassay for antibodies against Streptococcus pneumoniae "Abstr V-2, p617, 1994, A erican Society for Microbiology, Washington, D.C.). In addition, limited in vivo protection studies have shown that antibodies against the 37 kDa protein protect mice from lethal challenges. (Talkington et al., 1996 "Protection of rice agilest fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA)" Micro21.
[0004]
S. The gene encoding PsaA from pneumoniae strain R36A (non-encapsulated strain) was cloned into Escherichia coli and sequenced; however, this strain is highly conserved among various serotypes does not include the 37kDa protein encoded by the nucleic acid (Sampson et al., 1994, "Cloning and nucleotide sequence analysis of PsaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp.adhesins" Infect.Immun.62: 319 ~ 324). This particular nucleic acid and corresponding polypeptide are therefore limited in value for use as diagnostic reagents, in infection prevention, in infection treatment, or in vaccine development. US patent application Ser. No. 08 / 715,131, filed Sep. 17, 1996 (currently US Pat. No. 5,854,416, which is US patent application Ser. No. 08/222, filed Apr. 4, 1994). 179, which is now abandoned, which is a U.S. patent application Ser. No. 07 / 791,377 filed Sep. 17, 1991 (now U.S. Pat. No. 5,422). , 427), all of which are hereby incorporated by reference in their entirety), an isolated nucleic acid encoding a Streptococcus pneumoniae 37-kDa protein, a sample At least 10 nucleotides of this nucleic acid that can be used in a method for detecting the presence of Streptococcus pneumoniae in Specific fragments of tides, and immunogenic vaccine is disclosed. Further disclosed is a purified polypeptide encoded by this nucleic acid encoding the Streptococcus pneumoniae 37-kDa protein that can be used in an immunogenic vaccine. In addition, purified antibodies that bind to the 37-kDa protein of Streptococcus pneumoniae or fragments thereof (which can be used in methods of detecting the presence of Streptococcus pneumoniae, as well as in therapeutic and prophylactic methods) are disclosed. Sequence conservation is a necessary requirement for species-specific vaccine candidates. Among the types of pneumococci, and particularly among encapsulated pneumococci, sequence conservation of the PsaA gene, which results in many serious disease cases, is still under investigation.
[0005]
To provide a peptide that can serve as a vaccine against multiple strains of Streptococcus pneumoniae. There is a need to identify characteristic epitopes associated with pneumoniae PsaA. The present invention relates to S.I. This need is addressed by determining effective epitope peptides associated with pneumoniae PsaA and using these peptides in therapeutic compositions and vaccines against Streptococcus pneumoniae infections.
[0006]
(Summary of the Invention)
The present invention provides novel immunogenic peptides obtained from random libraries by screening for high affinity binding to monoclonal antibodies specific for the PsaA epitope. Furthermore, the peptides of the present invention serve as immunogens in a subject, thereby effectively inducing the production of antibodies by the subject, and in addition, S. It has the ability to confer protective immunity against infection by pneumoniae. The invention also relates to the selection method used to obtain such peptides.
[0007]
The peptide of the present invention is a residue, the sequence of which is the following: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments thereof Including peptides containing more selected residues. In certain embodiments, the peptide is essentially selected from the following: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments thereof. Consists of an array. In certain embodiments, the peptide consists of a sequence selected from: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments thereof.
[0008]
The present invention further provides a peptide as described above. Here, this peptide is a multi-antigenic peptide. In one embodiment, the multi-antigenic peptide comprises a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments thereof. Having at least one arm. In another embodiment, the multi-antigenic peptide consists essentially of a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments thereof. Having at least one arm. In another embodiment, the multi-antigenic peptide consists of a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments thereof. Having at least one arm.
[0009]
The multi-antigenic peptide can also be a bipeptide comprising any combination of SEQ ID NOs: 5-10, or immunogenic fragments thereof. In one embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 5 and at least one second arm comprising SEQ ID NO: 6. In another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 5 and at least one second arm comprising SEQ ID NO: 7. In another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 5 and at least one second arm comprising SEQ ID NO: 8. In another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 5 and at least one second arm comprising SEQ ID NO: 9. In another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 6 and at least one second arm comprising SEQ ID NO: 7. In another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 6 and at least one second arm comprising SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 10 and at least one second arm comprising SEQ ID NO: 9. In yet another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 10 and at least one second arm comprising SEQ ID NO: 7. In yet another embodiment, the multi-antigenic peptide has at least one first arm comprising SEQ ID NO: 5 and at least one second arm comprising SEQ ID NO: 10.
[0010]
The multi-antigenic peptide can also be a tripeptide comprising any combination of SEQ ID NOs: 5-10, or immunogenic fragments thereof. For example, in one embodiment, the multi-antigenic peptide comprises at least one first arm comprising SEQ ID NO: 5, at least one second arm comprising SEQ ID NO: 6, and at least one third arm comprising SEQ ID NO: 7. Has an arm. In another embodiment, the multi-antigenic peptide comprises at least one first arm comprising SEQ ID NO: 5, at least one second arm comprising SEQ ID NO: 9, and at least one third arm comprising SEQ ID NO: 10. Have. In still other embodiments, the multi-antigenic peptide has a first arm comprising SEQ ID NO: 5, a second arm comprising SEQ ID NO: 10, and a third arm comprising SEQ ID NO: 7, 8, or 9; or The multi-antigenic peptide has a first arm comprising SEQ ID NO: 5, a second arm comprising SEQ ID NO: 6, and a third arm comprising SEQ ID NO: 8 or SEQ ID NO: 9; or Having a first arm comprising number 6, a second arm comprising SEQ ID NO: 7, and a third arm comprising SEQ ID NO: 8, 9, or 10; or the multi-antigenic peptide comprises a first arm comprising SEQ ID NO: 7 , A second arm comprising SEQ ID NO: 8, and a third arm comprising SEQ ID NO: 9 or 10, etc.
[0011]
In any of the above embodiments, either arm can be an immunogenic fragment of SEQ ID NO: 5, 6, 7, 8, 9, or 10.
[0012]
In another aspect, the present invention is a peptide that immunospecifically binds to a monoclonal antibody obtained in response to immunization of an animal with Streptococcus pneumoniae PsaA, wherein the peptide is lipid solubilized. In one embodiment, the peptide is lipid solubilized with monopalmitic acid.
[0013]
The present invention further provides S. cerevisiae in a subject. a therapeutic composition, wherein the immunogenic peptide is combined with an immunostimulatory carrier to be administered to the subject to elicit an immune response that confers protective immunity against infection by pneumoniae, and / or Or provide a vaccine.
[0014]
The present invention further provides S. cerevisiae in a subject. Therapeutic compositions and / or vaccines in which the immunogenic peptide is combined with an adjuvant to be administered to the subject to elicit an immune response that confers protective immunity against infection by pneumoniae I will provide a.
[0015]
The present invention further provides therapeutic compositions and / or vaccines, wherein the immunogenic peptide is a multi-antigenic peptide of the present invention as described above.
[0016]
The invention further provides a therapeutic composition and / or vaccine wherein the immunogenic peptide is a peptide that immunospecifically binds to a monoclonal antibody obtained in response to immunization of an animal with Streptococcus pneumoniae PsaA. Provided (wherein the peptide is lipid solubilized). In one embodiment, the peptide is lipid solubilized with monopalmitic acid.
[0017]
The present invention still further provides S. cerevisiae in a subject. Describes a method of conferring protective immunity against infection by pneumoniae. Here, the therapeutic composition and / or vaccine of the present invention is administered to the subject.
[0018]
A further aspect of the present invention is an S.A. 2 shows a method for identifying a peptide that incorporates PsaA or a fragment thereof (ie, an immunogenic peptide) that elicits an immune response in a subject against pneumoniae. The method includes preparing a random peptide library, screening the peptide library to identify the immunogenic peptide, and obtaining an amino acid sequence of the immunogenic peptide.
[0019]
The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit the claimed invention. Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entirety and more fully describe the state of the art to which this application belongs.
[0020]
(Detailed description of the invention)
As used herein, an “immunogenic peptide” refers to a peptide that elicits an immune response when administered to a subject or otherwise ingested by a subject. An immune response includes at least the production of antibodies that specifically bind to an immunogenic substance (ie, a humoral response). The immunogenic substance can further elicit a cellular immunological response. Such immunogens are S. cerevisiae. Any of the immunogenic peptides obtained by screening a library of random peptides with a monoclonal antibody that immunospecifically reacts with PsaA from Pneumoniae.
[0021]
As used herein, “immune response” and “immunogenic response” can include at least a humoral response, ie, the production of antibodies that specifically bind to an immunogenic agent. An immunogenic response can refer to a cellular immunological response, either alternatively or additionally.
[0022]
As used herein, “protective immunity” means that a subject has produced antibodies (at least some of which are neutralizing antibodies) in response to exposure to a pathogen-related immunogen. State. The neutralizing antibody binds to the immunogenic component of the pathogen such that the productive infection by the pathogen is inhibited or eliminated, so that the subject is essentially free of symptomatic disease. Protective immunity can also be elicited from alternative immunogenic responses that lead to inactivation, loss or destruction of pathogen factors.
[0023]
As used herein, an “immunostimulatory carrier” refers to any of a variety of immunogenic biological polymers that, when introduced into a subject, themselves elicit an immune response. An immunostimulatory carrier, when used in combination with an immunogen of interest (eg, a peptide of the invention) provides an enhanced immunogenic response of the subject to the immunogen of interest. Further, as used herein, “adjuvant” refers to a composition that, when administered in combination with an immunogenic substance, enhances the immunogenic activity of that substance.
[0024]
As used herein, a “library” refers to a set of fragments derived from biopolymers, wherein each member of the set is a desired expression that expresses the desired biological function. A candidate for processing biological activity. The library is either a peptide library or a library of oligonucleotide fragments, each member comprising a nucleotide sequence that encodes a particular member of the peptide library. In the present invention, a peptide library is a set of peptides encoded by a random oligonucleotide library. The desired activity for a given peptide is that the peptide is S. aureus in the subject. It is immunogenic to pneumoniae.
[0025]
As used herein, a “subject” is a pathogen organism S. cerevisiae. A mammal in which it is desired to elicit an immune response against Pneumoniae. The main class of subjects of the present invention is S. cerevisiae. pneumoniae is actually a pathogenic human (especially infants and the elderly). Thus, in a human subject, the immune response is intended to be a protective immune response. For non-human mammals, see S. et al. pneumoniae may or may not be pathogenic in nature. Such non-human subjects used as laboratory animals that provide an immune response may be useful for the characterization and optimization of the compositions and methods of the invention. Such mammals include mice, rats, and non-human primates as non-limiting examples. A further class of subjects includes animals (including pets and livestock animals) provided for veterinary practice. S. If pneumoniae is pathogenic in such a subject, it is desirable to elicit protective immunity.
[0026]
“Purified protein” (purified protein), as used herein, relates to distinguishing proteins from contaminants or cellular components, where the protein or fragment is usually a contaminant in which the protein is present together. Or it means not containing enough cellular components. It is not intended that “purified (purified)” should have a preparation that is technically completely pure (homogeneous), but “purified (purified)” is used herein. When done, it means sufficiently separated from contaminants or cellular components that are normally present together to provide the protein in a state that can be used in an assay (eg, immunoprecipitation or ELISA). For example, purified protein can be used in an electrophoresis gel.
[0027]
As used herein, “stringent conditions” refers to the washing conditions used in a nucleic acid hybridization protocol. In general, washing conditions are such that the denaturation temperature is calculated for the nucleic acid hybrid under study. _m It should be a combination of temperature and salt concentration, selected to be about 5-20 ° C. below the value. Temperature and salt conditions are readily determined empirically in a preliminary experiment, in which a sample of reference DNA immobilized on a filter is relative to the protein encoding the probe or nucleic acid of interest. Hybridized and then washed under conditions of different stringency. T of such an oligonucleotide _m Values can be estimated by considering about 2 ° C. for each A or T nucleotide and about 4 ° C. for each G or C. Thus, for example, a 50% G-C 18 nucleotide probe yields an estimated T of 54 ° C. _m Has a value.
[0028]
As used herein, a “therapeutic composition” is a composition of the invention that can be administered to a subject to elicit a protective immune response, and alone or in combination with an immunostimulatory carrier or adjuvant. A composition comprising one or more of the immunogenic peptides. The therapeutic composition comprises the peptide and carrier, either as a mixture or as a chemical conjugate. Together they constitute an active agent. Where more than one peptide is used and the composition is a conjugate, each peptide can be either alone or conjugated to an immunostimulatory carrier. In addition, therapeutic compositions generally include components of a pharmaceutical formulation in which the active agent is suspended or dissolved. The components of pharmaceutical formulations are well known to those skilled in immunology or pharmaceutical science. The formulation should be appropriate to administer the active agent to the subject to elicit an immune response and to confer protective immunity against the pathogen associated with the immunogenic peptide.
[0029]
As used herein, the term "allelic variation" or "allelic variant" An immunogenic PsaA peptide or protein obtained from a pneumoniae serotype, other than a reference serotype (eg, serotype 2). Allelic variants describe similar proteins that diverged by less than 15% in their corresponding amino acid identity from the same 37 kDa pneumococcal surface adhesion protein, or 37 kDa Streptococcus pneumoniae protein shown in the sequence listing as SEQ ID NO: 2. Preferably, the allelic variant is less than 10% branched in its corresponding amino acid identity, more preferably less than 7% branched, more preferably less than 5% in its corresponding amino acid identity. Branches, more preferably less than 3% branches, more preferably less than 2% branches, and most preferably less than 1% branches. These amino acids can be substitutions within the amino acid sequence shown in the sequence listing as SEQ ID NO: 2 or the variant is deleted from the amino acid sequence shown in the sequence listing as SEQ ID NO: 2 or this amino acid sequence Can be added to.
[0030]
(Nucleic acid)
In one aspect, the present invention provides an isolated nucleic acid encoding a Streptococcus pneumoniae 37 kDa protein, the amino acid sequence of which is shown in the Sequence Listing as SEQ ID NO: 2. The term “isolated” refers to S. cerevisiae. A nucleic acid essentially isolated from other genes naturally present in Pneumoniae. In one embodiment, the present invention provides an isolated nucleic acid encoding a 37 kDa protein of Streptococcus pneumoniae, wherein the nucleic acid is a nucleic acid whose nucleotide sequence is shown in the sequence listing as SEQ ID NO: 1 . Also provided is an isolated nucleic acid comprising a unique fragment of at least 10 nucleotides of the nucleic acid shown in the sequence listing as SEQ ID NO: 1.
[0031]
“Unique fragment” as used herein means a nucleic acid of at least 10 nucleotides that is not identical to any other nucleic acid sequence known at the time the invention was made. Examples of sequences of at least 10 nucleotides that are unique to the nucleic acids shown in the sequence listing as SEQ ID NO: 1 are DNASIS (Hitachi Engineering, Inc.), for sequences listed on GenBank or for other sequence databases. Or using a computer program such as Word Search or FASTA from Genetics Computer Group (GCG) (Madison, WI) (which searches nucleotide sequences listed for similarity to the nucleic acid in question) It can be easily confirmed by comparing. A sequence is unique if it does not match any of the known sequences. For example, a sequence of nucleotides 1-10 can be used to search a database for identical matches. If no match is found, nucleotides 1-10 represent a unique fragment. The sequence of nucleotides 2-11 can then be used to search the database, then the sequence of nucleotides 3-12, and thus nucleotides 1321-1330 of the sequence shown in the sequence listing as SEQ ID NO: 1 Can be used to search up to. The same type of search can be performed on 11 nucleotide, 12 nucleotide, 13 nucleotide sequences, and the like. Possible fragments range from 10 nucleotides in length to 1 nucleotide less than the sequence set shown in the sequence listing as SEQ ID NO: 1. These unique nucleic acids, as well as degenerate nucleic acids, can be used, for example, as primers for amplifying nucleic acids from other strains of Streptococcus pneumoniae to isolate allelic variants of the 37 kDa protein or 37 kDa protein It can be used as a primer for reverse transcripts of RNA or can be used as a probe for use in detection techniques such as nucleic acid hybridization. Those skilled in the art will recognize that even if a nucleic acid of at least 10 nucleotides is unique to a particular gene, the nucleic acid fragment can still hybridize to many other nucleic acids and is therefore used in techniques such as amplification and nucleic acid detection. Recognize that you get.
[0032]
As shown in the sequence listing as SEQ ID NO: 2, Nucleic acids encoding allelic variants of the 37 kDa protein of pneumoniae are also provided. The homology between the protein coding regions of the nucleic acid encoding an allelic variant of the 37 kDa protein is preferably with less than 20% branching from that region of the nucleic acid shown in the sequence listing as SEQ ID NO: 1 encoding the 37 kDa protein. is there. Preferably, the corresponding nucleic acids are less than 15% branched in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 10% branched in their sequence identity, more preferably less than 7% branched, more preferably less than 5% in their corresponding nucleotide identity. Branches, more preferably less than 4% branches, more preferably less than 3% branches, more preferably less than 2% branches, and most preferably less than 1% branches. In particular, nucleic acid variations can be made from the protein shown in the sequence listing as SEQ ID NO: 2 up to about 15% amino acid sequence variation.
[0033]
One skilled in the art will recognize that a nucleic acid encoding a homologue or allelic variant of the 37 kDa protein shown in the sequence listing as SEQ ID NO: 2 can be isolated from related Gram positive bacteria. Nucleic acid encoding a 37 kDa protein can be obtained by any of a number of techniques known to those of skill in the art. Methods for isolating nucleic acids of the invention, including probes and primers that can be used, are described in US patent application Ser. No. 08 / 715,131 (filed Sep. 17, 1996, now US Pat. No. 5,854,416). No. 08 / 222,179 (filed Apr. 4, 1994, now abandoned, which is abandoned by U.S. Patent Application No. 07 / 791,377 (September 17, 1991). Application, now partly pending in US Pat. No. 5,422,427). General methods that can be utilized for these purposes include: Sambrook et al. “Molecular Cloning: a Laboratory Manual” Cold Spring Harbor Laboratory Press (1989), and Ausubel et al. “Current Protocols in Molecular Wholesole in Health. York, 1987 (revised quarterly). Amplification procedures that can be used in nucleic acid isolation protocols (eg, amplification using the polymerase chain reaction (ie, “PCR”)) are well known to those skilled in the art (eg, Innis et al., 1990 “PCR Protocols: A Guide to See Methods and Applications, Academic Press, Inc.). An example of amplification of a nucleic acid encoding a 37 kDa protein of Streptococcus pneumoniae serotype 6B is discussed in the Examples included herein.
[0034]
(37 kDa protein (PsaA))
The invention also provides a purified polypeptide shown in the sequence listing as SEQ ID NO: 2, as well as a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO: 1. This protein can be used as a vaccine composition as well as a reagent to identify a subject's antibody raised against Streptococcus pneumoniae during infection. The purified protein can also be used in a method for detecting the presence of Streptococcus pneumoniae.
[0035]
Unique fragments of the 37 kDa protein can be identified in the same manner that is used to identify unique nucleic acids. For example, a protein sequence database can be searched using sequences of 3 amino acids or more derived from the sequence of a 37 kDa protein as shown in the sequence listing as SEQ ID NO: 2. Therefore, sequences that do not match known sequences are unique. A method for preparing these proteins and protein fragments is described in US patent application Ser. No. 08 / 715,131 (filed Sep. 17, 1996, now US Pat. No. 5,854,416, which is incorporated herein by reference). No. 08 / 222,179 (filed Apr. 4, 1994, now abandoned, which was issued in U.S. Patent Application No. 07 / 791,377 (filed Sep. 17, 1991, now U.S. Pat. , 422, 427), which is partly dependent).
[0036]
The present invention provides peptide fragments related to the 37 kDa pneumococcal surface adhesion protein. A polypeptide fragment of the invention is a recombinant polypeptide obtained by cloning a nucleic acid encoding a polypeptide fragment in an expression system capable of producing the polypeptide fragment, as described above for the 37 kDa protein. obtain. For example, an immunoreactive peptide associated with a 37 kDa pneumococcal surface adhesion protein that can cause a significant immune response is cloned using an antibody raised against the adhesion protein, and the nucleic acid encoding the polypeptide is cloned into an expression vector; And can be identified by isolating that particular polypeptide for further use (eg, diagnosis, therapy, and vaccination). Amino acids that do not contribute to immunoreactivity and / or specificity can be deleted without loss in their respective activities. For example, the amino-terminal amino acid or carboxy-terminal amino acid can be sequentially removed from any peptide identified using the procedure outlined above, and any of the many available assays with immunoreactivity. Tested in. Alternatively, internal amino acids can be removed sequentially and immunoreactivity is tested for each deletion.
[0037]
In another example, a peptide fragment associated with a 37 kDa pneumococcal surface adhesion protein comprises at least one amino acid comprising a specific portion or any portion of an amino-terminal amino acid or a carboxy-terminal amino acid, or further an internal region of a polypeptide. A modified polypeptide may be substituted in place of the originally occupied amino acid residue and replaced with a polypeptide fragment or other moiety (eg, biotin that may facilitate purification of the modified 37 kDa pneumococcal surface adhesion protein).
[0038]
Immunoreactive peptide fragments associated with the 37 kDa pneumococcal surface adhesion protein include insertions of specific regions or specific amino acid residues as long as the peptide immunoreactivity is not significantly impaired compared to the 37 kDa pneumococcal surface adhesion protein. , Deletions, substitutions or other selected modifications. These modifications may provide a number of additional properties, such as, for example, to remove / add amino acids that can disulfide bond, to increase their lifetime. In any case, the peptide must possess bioactive properties (eg, immunoreactivity) comparable to the 37 kDa pneumococcal surface adhesion protein.
[0039]
(antibody)
The present invention uses a purified antibody that selectively binds to a polypeptide encoded by the nucleic acid shown as SEQ ID NO: 1 in the sequence listing or a polypeptide encoded by a unique fragment of at least 10 nucleotides of SEQ ID NO: 1. . The antibody (either polyclonal or monoclonal) can be raised against its naturally occurring form or recombinant form thereof, the 37-kDa pneumococcal surface attachment factor protein or a unique fragment thereof. The antibody can be used in various techniques or procedures (eg, diagnosis, treatment or immunity). Antibodies are prepared by a number of well-known methods (see, eg, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1988)). Briefly, purified antigen can be injected into an animal in an amount and at an interval sufficient to elicit an immune response. The antibody can be purified directly to obtain a polyclonal antibody. Alternatively, spleen cells can be obtained from this animal. The cells can then be fused with an immortal cell line and screened for antibody secretion to obtain monoclonal antibodies. This antibody can be used to screen a nucleic acid clone library for cells secreting the antigen. These positive clones can then be sequenced (see, for example, Kelly et al., Bio Technology, 1992, 10: 163-167: Bebbington et al., 1992 Bio Technology, 10: 169-175).
[0040]
The phrase “selectively binds” to a polypeptide refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, a specified antibody bound to a particular protein does not bind in a significant amount to other proteins present in the sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies that selectively bind to a particular protein. For example, solid phase ELISA immunoassays are routinely used to select antibodies that selectively immunoreact with proteins. See Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that can be used to select for selective binding. In some instances, it may be desirable to prepare monoclonal antibodies from various subjects. A description of techniques for preparing such monoclonal antibodies can be found in Stites et al., “Basic and Clinical Immunology” (Lange Medical Publications, Los Altos, Calif., 4th edition) and references cited therein. As well as in Harlow and Lane ("Antibodies: A Laboratory Manual", "Cold Spring Harbor Publications, New York, (1988)).
[0041]
Monoclonal antibodies (MAbs) used in the present invention (US patent application Ser. No. 08 / 715,131, filed Sep. 17, 1996, now incorporated by reference); 854, 416)) are fragments of MAb 1E7A3D7C2 or antibody 1E7A3D7C2 that retain characteristics (eg, their binding specificity and binding affinity); MAb 1B6E12H9 or fragments that retain characteristics of antibody 1B6E12H9 MAb 3C4D5C7 or a fragment thereof retaining the characteristics of antibody 3C4D5C7; MAb 4E9G9D3 or a fragment thereof retaining the characteristics of antibody 4E9G9D3; MAb 4H5C10F3 or a fragment thereof retaining the characteristics of antibody 4H5C10F3 ; A fragment thereof that retains the characteristics of and MAb 8G12G11B10 or antibodies 8G12G11B10; fragments thereof retaining the characteristic of MAb 6F6F9C8 or antibody 6F6F9C8.
[0042]
Hybridomas used to produce the respective monoclonal antibodies used in the present invention (US patent application Ser. No. 08 / 715,131, filed Sep. 17, 1996, incorporated herein by reference) (Disclosed in US Pat. No. 5,854,416) are hybridoma 1E7A3D7C2, hybridoma 1B6E12H9, hybridoma 3C4D5C7, hybridoma 4E9G9D3, hybridoma 4H5C10F3, hybridoma 6F6F9C8 and hybridoma 8G12G11B10.
[0043]
(Therapeutic composition)
Also provided by the present invention is a therapeutic composition comprising an immunogenic polypeptide encoded by the nucleic acid shown as SEQ ID NO: 1 in the sequence listing or a unique fragment of at least 10 nucleotides of SEQ ID NO: 1. The invention also provides a therapeutic composition comprising at least one immunogenic peptide that immunospecifically binds to a monoclonal antibody obtained in response to immunizing an animal with Streptococcus pneumoniae PsaA. Examples of such immunogenic peptides are shown in SEQ ID NO: 5 to SEQ ID NO: 10. This therapeutic composition is preferably combined with an immunostimulatory carrier. The therapeutic composition, when administered to a subject, is S. cerevisiae. Protective immunity against pneumoniae infection.
[0044]
For protection against specific diseases, infections or conditions caused by organisms that have induced 37-kDa pneumococcal surface adhesion factor protein (or unique fragments thereof) using the polypeptides provided by the present invention, Subjects can be vaccinated. Serotype 6B 37-kDa Streptococcus pneumoniae surface adhesion factor protein polypeptide or a unique fragment thereof is used to inoculate a subject organism so that the subject is later infected by the polypeptide-derived organism. The subject can generate an active immune response against the presence of the polypeptide or polypeptide fragment that can be protected from. Those skilled in the art can provide an immune response (especially a cell-mediated immune response) to a 37-kDa pneumococcal surface adhesion factor protein from a particular strain that can later provide protection from reinfection or infection by related strains. Recognize However, the 37-kDa protein provided by the present invention is S. cerevisiae. It is relatively conserved among the 90 serotypes of Pneumoniae and can therefore serve as a multivalent vaccine. Immunization with a 37-kDa pneumococcal surface adhesion factor protein or immunogenic peptide of the present invention can be accomplished by administering 37-kDa pneumococcal surface adhesion factor protein to a subject alone or pharmaceutically acceptable. It can be achieved by either administering to a subject with a carrier (Kby, J. 1992 “Immunology”, WH Freeman and Co., New York). The immunogenic amount of a 37-kDa pneumococcal surface adhesion factor protein or immunogenic peptide of the invention can be determined using standard procedures.
[0045]
Briefly, various concentrations of a polypeptide of the invention are prepared, administered to a subject, and an immunogenic response to each concentration (eg, production of antibodies or cell-mediated immunity to the polypeptide) is determined. . Techniques for monitoring both cellular and humoral immunogenic responses of patients following inoculation of this polypeptide are well known in the art. For example, samples are assayed using an enzyme linked immunosorbent assay (ELISA) to identify specific antibodies (eg, serum IgG (Hjelt et al., J; Med. Virol., 21: 39-47, (1987))). Presence of lymphocytes or cytokines can also be monitored. The specificity of the putative immunogenic antigen of any particular polypeptide can be determined by sera, other fluids or lymphocytes from the inoculated patient, other closely related 37-kDa pneumococcal surface attachment factor proteins. Can be confirmed by testing for cross-reactivity. The amount of the 37-kDa pneumococcal surface adhesion factor polypeptide or immunogenic peptide of the invention administered is immunized, depending on the subject, the condition of the subject, the size of the subject, etc. It is at least an immunogenic amount for a particular subject. The polypeptide can be formulated with a pharmaceutically acceptable carrier, with an adjuvant, and with additional compounds (including cytokines).
[0046]
Pharmaceutically acceptable carriers or adjuvants in the therapeutic compositions of the present invention are standard criteria (Arnon, R. (eds.) “Synthetic Vaccines”, I: 83-92, CRC Press, Inc., Boca Raton. , Florida, 1987). By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable (ie, the material is any of the other components of the pharmaceutical composition containing it). And can be administered to an individual with a selected compound without causing any undesirable biological effects or interacting in an undesirable manner). The carrier or adjuvant can depend on the mode of administration and the particular patient. The method of administration can be performed parenterally, orally, sublingually, mucosally, by inhalation, by absorption, or by injection. Actual methods of preparing suitable dosage forms are well known or apparent to those skilled in the art: For example, Remington's Pharmaceutical Sciences (Martin, EW (ed.), Latest edition, Mack Publishing Co., See Easton, PA). When used, parenteral administration is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or emulsions. Another approach for parenteral administration involves the use of a slow release system or a sustained release system so that a constant level of dosage is maintained (eg, US Pat. No. 3, 710, 795). In addition, powders or aerosols can be formulated for administration by inhalation.
[0047]
(Detection method)
The present invention provides a method for detecting the presence of Streptococcus pneumoniae in a subject based on several variations of an immunoassay, the method comprising a purified poly-protein encoded by the nucleic acid shown as SEQ ID NO: 1 in the sequence listing. Peptide, purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO: 1, selectively purified polypeptide encoded by a nucleic acid shown as SEQ ID NO: 1 in the sequence listing Using either an antibody or an antibody that selectively binds a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO: 1 and binding of the antibody to the polypeptide This binding detects FIG. 3 shows the presence of Streptococcus pneumoniae in a subject. There are numerous immunodiagnostic methods that can be used to detect antigens or antibodies, as exemplified by the following non-inclusive examples. These methods as well as other methods can not only detect the presence of an antigen or antibody, but can also quantify the antigen or antibody. These methods are described in US patent application Ser. No. 08 / 715,131 (filed Sep. 17, 1996 (currently US Pat. No. 5,854,416), US patent application Ser. No. 08 / 715,131 US patent application Ser. No. 08 / 222,179, which is a continuation-in-part of US application Ser. No. 08 / 222,179, filed Apr. 4, 1994 (currently abandoned). 791,377 (shown in Sep. 17, 1991 (currently a continuation-in-part of US Pat. No. 5,422,427). Generally, detection methods that can be used in the practice of the present invention are: For example, Harlow et al., “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor. , New York, (1988).
[0048]
(Infection treatment and prevention methods)
The present invention also includes S.I. Provided is a method of preventing Streptococcus pneumoniae infection in a subject at risk of infection by pneumoniae, the method being encoded by a nucleic acid encoding a Streptococcus pneumoniae 37-kDa protein, as shown in the sequence listing as SEQ ID NO: 1. Or an immunogenic polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO: 1, or an immunogenic peptide of the invention (e.g., SEQ ID NO: 5 to SEQ ID NO: 10) comprising administering an effective amount of a therapeutic composition comprising the immunogenic peptide (10) alone or in combination with a pharmaceutically acceptable carrier to the subject.
[0049]
The present invention further provides a method of treating Streptococcus pneumoniae infection in a subject, comprising: a polypeptide encoded by a nucleic acid as set forth in the sequence listing as SEQ ID NO: 1 or at least 10 nucleotides unique of SEQ ID NO: 1 Administering to the subject an effective amount of an antibody against a polypeptide encoded by a nucleic acid comprising a fragment of either alone or in combination with a pharmaceutically acceptable carrier. It is well known in the art to treat a subject already infected with a particular organism by administering an antibody against the organism to the subject. For example, immunoglobulin isolated from animals or humans previously exposed to rabies virus is currently a treatment for rabies virus infection. Better treatment of infected individuals can be achieved by administering monoclonal antibodies to these individuals. This is because these monoclonals react or bind more specifically than polyclonal (see, eg, Kaplan et al., “Rabies”, Sci. Am. 242: 120-134 (1980)).
[0050]
(Epitope immunogenic peptide)
The present invention relates to a novel epitopic immunogenic peptide obtained as a peptide encoded by a random oligonucleotide library by selecting for high affinity binding of the peptide by a monoclonal antibody specific for the epitope on the PsaA antigen Is disclosed.
[0051]
In a method known as “biopanning” or “panning”, the target protein or peptide is selected from a library that is expressed as a heterologous insert on the outer surface of the microorganism. For example, a bacterium or virus may have a nucleotide sequence encoding a heterologous peptide or protein sequence that is incorporated into its chromosomal nucleic acid in such a way that a fusion or chimera is created. This fusion represents the natural protein of the microorganism directly linked to the heterologous peptide or protein. Once expressed on the surface of the microorganism, it can be probed with a ligand specific to the required peptide or protein (eg, an antibody that recognizes a particular epitope). Once identified by capture, this heterologous sequence of either nucleic acid or protein can be obtained and identified.
[0052]
The normal performance of this procedure is well known to those skilled in the fields of protein chemistry, immunology and virology. Filamentous bacteriophages (eg, M13, fl or fd) are used. These bacteriophages have two well-known structural proteins on their surface: gene III protein and gene VIII protein. The phage nucleic acid is altered by incorporating a heterologous peptide fusion sequence in-frame with the gene for one or the other of these structural proteins. When seeking a target peptide from such a large set of peptides (ie, a library), a corresponding library of heterologous nucleotide sequences encoding members of the peptide library is incorporated into the structural protein gene. The resulting bacteriophage population (referred to as a phage display library) is a procedure that optimizes the selection of only viral particles that express members of this peptide library with high affinity for PsaA specific ligands (eg, MAbs). Provided. The bacteriophage particles thus selected can then be amplified by further culture, or their nucleic acids can be amplified by methods such as the polymerase chain reaction. In this way, the nucleic acid of the captured particles can be isolated and sequenced to provide the coding sequence for the high affinity epitope bound to the MAb or other ligand. Biopanning is described in, for example, Smith, G. et al. P. And K.K. K. Scott (1993, “Libraries of Peptides and Proteins Displayed on Filamentous Page”, Meth. Enzymol. 217: 228-257).
[0053]
The immunogenic peptides of the present invention were obtained using a biopanning procedure with general applicability to identify peptide sequences that could potentially elicit protective immunity against pathogenic microorganisms. This method includes the following steps:
(A) providing a library composed of random oligonucleotides, wherein the oligonucleotides are about 30-45 nucleotides in length;
(B) the coat protein in codons and in-frame with the amino acid residues of the coat protein so that the gene for the coat protein of the filamentous bacteriophage is contained within the complete nucleic acid that is the genome for the bacteriophage. The antibody can bind by inserting a library oligonucleotide into the gene for and exposing it to the surface when this coat protein is expressed and incorporated into a complete bacteriophage particle Placing the oligonucleotide in the gene so that the peptide is available as an epitope, thereby creating a bacteriophage library;
(C) increasing the bacteriophage library carrying the oligonucleotide library by culturing the bacteriophage library in a host that can be infected by the bacteriophage;
(D) any bacteriophage particle that immunospecifically reacts with a monoclonal antibody obtained upon immunization of an animal with an immunogen (eg, an immunogenic peptide or immunogenic polypeptide) from a pathogenic microorganism Screening this expanded bacteriophage library for; and
(E) sequencing the gene for the coat protein of any bacteriophage particle obtained in step (d), whereby the translation product has a sequence of peptides that can potentially elicit protective immunity against Streptococcus pneumoniae Generating the nucleotide sequence of that member of the oligonucleotide library.
[0054]
For the particular application used in obtaining the immunogenic peptides of the present invention, the above method is described in S.H. For Pneumoniae, the coat protein is a gene III protein that is a filamentous bacteriophage tail protein (eg, M13, fl or fd), and a monoclonal antibody immunizes an animal with Streptococcus pneumoniae pneumococcal surface adhesion A protein (PsaA). Is obtained according to what you do. This peptide is obtained using a procedure that emphasizes thinning only peptides with high affinity for this antibody. This increases the likelihood that the selected peptide will effectively induce a protective humoral immune response.
[0055]
The sequence of this peptide that binds to this antibody can be identified by sequencing the gene III fusion of bacteriophage particles obtained in the biopanning process. The actual immunogenic peptide can then be synthesized using a conventional peptide synthesizer. These peptides are then incorporated into a therapeutic composition in which the immunogenic peptide is combined with an immunostimulatory carrier that is administered to the subject. Once administered in an effective amount, the subject is treated with S. cerevisiae. Induces production of antibodies against pneumoniae. This is because S.A. The subject is conferred with protective immunity against infection by pneumoniae.
[0056]
PsaA is effectively immunogenic, S. cerevisiae. It is a 37-kDa species-common protein derived from pneumoniae (pneumococcus). This is common to all serotypes whose polysaccharide is a component of the currently used pneumococcal vaccine (Russell et al., 1990, “Monoclonal Anti-recognizing a specifications-specific protein from Streptococcus. Microbiol. 28: 2191-2195). The sequence of the PsaA gene cloned from serotype R36A has been described (US Pat. No. 5,422,427 to Russell et al.) And the sequence of the PsaA protein was deduced. In addition, the nucleotide sequences of cloned PsaA from serotype 2 and serotype 6B, and their corresponding amino acid sequences have been determined (Berry et al., 1996, “Sequence heterogeneity of PsaA, a 37-kilodalton putative essensessence. for vigorence of Streptococcus pneumoniae, Infect Immun. 64: 5255-5262; Sampson et al., 1997 1971). Except the putative leader sequence, there are 6 amino acid differences between PsaA from serotype 6B and serotype 2 from a total of 290 residues; between 6B and 36A Among them, there are 45 amino acid differences (Sampson et al., Ibid). From this result, Sampson et al. Suggested that serotype 2 and serotype 6B represent prototype sequences of the pneumococcal PsaA protein. PsaA from serotype 3 (disclosed in US patent application Ser. No. 08 / 715,131 (now US Pat. No. 5,854,416), incorporated herein by reference) and serotype 22 Derived from PsaA (Talkington et al., 1996, “Protection of rice Against Fatalpneumococcal Challenge by Immunization with pneumococcal surface adhesin A (PsaA). Effectively provides protective immunity in mice against challenge doses of pneumoniae.
[0057]
The peptides of the present invention comprise an immunogenic epitope selected by binding to a PsaA specific monoclonal antibody. Preferably, the peptide is about 10-25 amino acid residues long. More preferably, the peptide is about 12-22 amino acid residues long, and most preferably about 15 amino acids long. In the embodiments shown in the examples below, the peptides of the invention have the sequences provided in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. Furthermore, the present invention includes immunogenic peptides that may be shorter than these sequences. Thus, for example, an immunogenic fragment of SEQ ID NO: 5, an immunogenic fragment of SEQ ID NO: 6, an immunogenic fragment of SEQ ID NO: 7, and an immunogenic fragment of SEQ ID NO: 8 are also encompassed by the present invention.
[0058]
Currently, S.M. About 90 serotypes of pneumoniae have been identified; these can have a PsaA antigen that is an allelic variant of the previously identified PsaA sequence. Thus, the present invention relates to allelic immunogenic peptides obtained, for example, by biopanning procedures in which monoclonal antibodies are raised by immunization with allelic variants, or by other methods known to those skilled in the relevant art. Include. The sequence of such a peptide is at least 80% identical to any of the following sequences; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, an immunogenic fragment of SEQ ID NO: 5, SEQ ID NO: 6 An immunogenic fragment of SEQ ID NO: 7, and an immunogenic fragment of SEQ ID NO: 8.
[0059]
The monoclonal antibody (MAb) disclosed above was further used in the procedure of the present invention. The specific MAbs used are named 1E7 (1E7A3D7C2), 6F6 (6F6F9C8), 4E9 (4E9G9D3), 8G12 (8G12G11B10), and 1B6 (1B6E12H9). These MAbs were obtained as a result of immunization of animals with PsaA; thus, such antibodies represent molecules whose antigen binding domain binds to an immunogenic epitope of the present invention.
[0060]
Identification of immunogenic epitopes associated with PsaA can be accomplished in any of a number of ways. Methods for identifying immunogenic epitopes may use any MAb obtained in response to primary immunization with PsaA. Any procedure that narrows the overall molecular structure of PsaA to a portion or fragment thereof can be used in identifying the immunogenic epitope. In one method, chemical modification of specific residues of PsaA results in a modified product that can impair reactivity with ligands such as anti-PsaA MAbs. Using knowledge of which residue (s) have been altered in the product with impaired binding, these residues can potentially be identified as part of an epitope. Furthermore, the biopanning described above can be used.
[0061]
In another method, a fragment of PsaA can be chemically synthesized by peptide synthesis. In general, a set of peptides is synthesized that represents a systematic progression along the entire protein sequence from its N-terminus to its C-terminus. An existing length window can be “walked” along the protein sequence to create a set of peptides that encompass most or all of the original sequence. Methods of peptide synthesis are well known to those skilled in the art of peptide chemistry, protein chemistry, and immunology. Once the peptide sequences are identified, commercially available equipment is available for their automated synthesis. The set of peptides obtained in this way can be subjected to an assay to ascertain whether they bind to PsaA specific ligands (eg, anti-PsaA MAbs). Immunoassay methods are preferred for such determinations and are well known to those skilled in immunology. These include procedures such as, for example, enzyme linked immunosorbent assays (ELISA), using competitive formats or direct heterogeneous formats. Peptides found to bind to PsaA specific ligands with high affinity are presumed to contain or include immunogenic epitopes of PsaA.
[0062]
The immunogenic peptides of the invention are identified by selection or screening procedures as described in the preceding paragraph. Next, a sequence of positively selected peptides needs to be obtained. In the case of chemical modification, the position of the inhibitory modification in the sequence results in a peptide centered on or including this modified residue. In the case of synthesized peptide screening, the sequence is readily available due to the identity of the positive sample. In the case of biopanning, positive bacteriophages are isolated and the nucleic acid is amplified either by expansion of phage particles in the culture or by amplification of the nucleic acid itself. The nucleic acid is then isolated and sequenced to identify the coding sequence for the heterologous peptide, and the coding sequence is translated to yield a peptide sequence.
[0063]
Once the sequence is known, the corresponding peptide is synthesized to function as an immunogenic peptide in the subject. Methods for synthesizing peptides are well known to those skilled in immunochemistry, immunology, and / or protein chemistry. For example, peptides can be obtained from Stewart et al., “Solid peptide synthesis”, 2nd edition, Pierce Chemical Co. , Rockford, IL (1984) and can be synthesized using solid phase F-moc chemistry. Typically, such synthesis is performed using an automated peptide synthesizer, such as an automated synthesizer available from Advanced ChemTech (Advanced ChemTech, Inc., Louisville, KY). An example of a synthesizer that can be used to synthesize peptides in accordance with the present invention is the Advanced ChemTech ACT model 396MPS. Once synthesized, the sequence is typically verified using an automated peptide sequencer such as the Porton model 2090 (Beckman Instruments Inc., Mountain View, CA).
[0064]
As demonstrated in the examples below and discussed in the “37 kDa protein” section above, the peptide immunoreactivity was not significantly impaired compared to the 37 kDa pneumococcal surface adhesion protein. The peptides of the present invention can include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues. With respect to the peptides of the present invention, the phrase “consisting essentially of” is intended to cover such modified peptides that can be identified and routinely made by those skilled in the art, as shown in the Examples section below. Is done.
[0065]
In some embodiments, the peptides of the invention are well known to those of skill in the art of immunochemistry and immunology and, as discussed herein in the section “Therapeutic compositions”, Prior to administration, it is combined with an immunostimulatory carrier and / or an adjuvant. In common practice, the immunostimulatory carrier is a protein such as keyhole limpet hemocyanin, bovine serum albumin, thyroglobulin, diphtheria toxins, and the like. The immunogenic peptide and carrier can be combined non-covalently or covalently. When combined non-covalently, they are mixed together so as to include the ingredients in the therapeutic composition administered to the subject.
[0066]
Numerous adjuvants are known in the art that can be used to stimulate an immune response against the peptides of the present invention. For example, alum, proteosome, specific lipid (eg, palmitic acid (see below)), QS21, or alhydrogel (2%; # A1090BS, Accurate Chemical and Scientific Company, Westbury, NY) It can be used as an adjuvant (da Fonseca, DP, et al., “Identification of new cytotoxic T-cell epitopes on the 38-kilodalton lipoproteins of mycobacterium. Sheikh, N .; Et al., "Generation of antigen specific CD8 + cytotoxic T cells following immunization with soluble protein formulated with novel glycoside adjuvants" Vaccine 17:. 2974 (1999); and Moore, A et al., "The adjuvant combination monophosphoryl lipid A and QS21 switches T cell responses induced with a soluble recombinant HIV protein from Th2 to Th1 "Vaccine 17: 2517 (1999)).
[0067]
In addition to the conjugates and adjuvants described above, the immunogenicity of the peptides of the invention can be enhanced by the addition of peptides to the proteosome or by the addition of cysteine residues. For addition to the proteosome, a spacer (eg, a CYGG spacer) and a lauroyl group can be added to the amino terminus of the peptide (Bio-Synthesis, Lewisville, TX). The lauroyl group enhances the hydrophobic complexation of peptide groups to the proteosome (Lowell, GH et al., “Proteosomes, hydrophobic anchors, iscoms, and liposomes for imprinted in N , Woodrow, GM, Levine, MM (eds.), Markel Dekker, Inc., New York, pages 141-160 (1990); Lowell, GH, et al., “Peptides bound to proteosomes visophies biosciences. feet become high immunogenic w "Thoth adjuvants" J. Exp. Med. 167: 658 (1988); and Zollinger, WD et al., "Complex of meningococcal group B polysaccharide and type 2 in mm lane. 836 (1979)). On the other hand, cysteine groups (eg, cysteine in the above CYGG spacer) enhance the immunogenicity of the peptide (Lowell, GH et al. (1990)). Proteosomes can be prepared from outer membrane complex vesicles from Group B Neisseria meningitidis 99M strain, as described by Zollinger (Zollinger et al., (1990)). Synthetic lipopeptides can be complexed to the proteosome in a 1: 1 (w / w) ratio by combining the components in the presence of a surfactant. Surfactants can be removed by exhaustive dialysis (Lowell, GH et al. (1988)).
[0068]
In some embodiments, the peptides of the invention are lipid solubilized with, for example, but not limited to, monopalmitic acid (Verhaul et al., “Monopalmic acid-peptide conjugates inducements” to stimulate an immune response against the peptide. cytotoxic T cell responses aginal marital epitopes: importance of spacer amino acids, J. Immunol. Methods, 182: 219 (1995)). A lipid-solubilized version of the peptide of the present invention containing monopalmitic acid deprotected palmitic acid (Sigma Chemicals, St. Louis, MO) using the same reaction conditions as the standard amino acid linkage described above. It can be synthesized by attachment to the amino terminus (Verhaul et al., Supra). In some embodiments, the triphasic peptide cysteine-serine-serine is added to the amino terminus of the peptides of the invention to facilitate the addition of lipids such as monopalmitic acid.
[0069]
In addition to adding lipids to the in vitro synthetic peptides of the present invention, lipid solubilized versions of the peptides of the present invention can be generated using recombinant DNA techniques using methods known to those skilled in the art. For example, a construct comprising a heterologous leader sequence linked to a nucleic acid encoding a peptide of the invention can be developed. Heterologous leader sequences are solubilized by the host organism. The leader sequence can be derived, for example, from the Borsperia burgdorferi ospA gene (Ades et al., “Recombinant lipidated PsaA protein, methods of preparation and use” PCT publication WO 99/40200 (1999)).
[0070]
The therapeutic compositions of the present invention are described in the “Therapeutic Composition” section above. In preparing the therapeutic compositions of the invention, the immunogenic peptide is formulated with a pharmaceutically acceptable vehicle for administration to a subject. Such vehicles are well known to those of ordinary skill in the pharmaceutical sciences and are administered by oral, sublingual or parenteral routes including but not limited to intravenous, subcutaneous, intramuscular, mucosal, and inhalation. Including preparations in liquid, gel, or solid form. These dosage forms can be conventional preparations such as solutions or suspensions with immediate bioavailability, or they slowly release the active immunogenic peptide over time It can be a controlled release formulation or controlled release device with properties. In a preferred embodiment, therapeutic compositions comprising the peptides of the present invention are administered S. cerevisiae in a subject to which they are administered, preferably a human subject. Provides protective immunity against Pneumoniae.
[0071]
In addition to the peptides disclosed by the methods described herein, immunogenic fragments of such peptides are also encompassed within the invention. An immunogenic fragment is any peptide shorter than the parent peptide whose sequence is identical to that of the portion of the peptide (parent) from which it is derived and which retains immunogenicity. It is generally understood in the field of immunochemistry that such peptides must be at least about 6 residues long in order to be antigenic. Thus, any fragment should be at least 6 residues long and have a maximum length that is 1 residue shorter than the parent peptide. Identifying immunogenic fragments can be accomplished using any method that confirms immunogenicity. These methods include, for example, the biopanning procedure described above, as well as combining an immunostimulatory carrier and a candidate peptide to form an active ingredient of the pharmaceutical composition, administering the pharmaceutical composition to a subject, and There is a direct demonstration of immunogenicity by assessing whether an immunogenic response has occurred.
[0072]
Peptide fragments that are specifically identified as being immunogenic can also be evaluated for their ability to elicit protective immunity in a subject. This is because experimental subject animals exhibiting an immunogenic response to the PsaA peptide fragment are S. cerevisiae. This is done using the methods described herein for determining whether to resist a challenge by pneumoniae.
[0073]
In some embodiments, the peptides of the invention are administered in combination with each other to enhance the effect of immunity. “In combination with” administration includes simultaneous and sequential administration as well as combined or separate administration. For example, in addition to a therapeutic composition in which the active agent is a single immunogenic peptide of the invention, the composition comprises SEQ ID NO: 5 or an immunogenic fragment thereof, SEQ ID NO: 6 or an immunogenic fragment thereof, SEQ ID NO: 7 or an immunogenic fragment thereof, SEQ ID NO: 8 or an immunogenic fragment thereof, SEQ ID NO: 9 or an immunogenic fragment thereof, SEQ ID NO: 10 or an immunogenic fragment thereof, or 10 to 25 residues in length, preferably It may include multiple peptides having the sequence given by the fragment of SEQ ID NO: 2, which is 12-22 residues, or more preferably about 15 residues. In one embodiment, the composition comprises a peptide having the sequence given by SEQ ID NO: 5 and a peptide having the sequence given by SEQ ID NO: 6. In another embodiment, the composition comprises a peptide having the sequences given by SEQ ID NO: 5 and SEQ ID NO: 9.
[0074]
(Multiple antigenic peptide)
In another embodiment for administering a peptide of the invention, the peptide is administered as a multiple antigenic peptide (MAP) (FIGS. 1-4). Methods for synthesizing and administering multi-antigenic peptides are known in the art (see, eg, Reynolds et al., “T and B epitopic degeneration and analysis of multiple peptides and the scientosomastosome samposoma samposoma samposome sampiosome sapiens samposom samposom espo mess ent esmo mess esmo sampos omos espo s es mer smo s es s om s ε e s s s e n om ε iε m e t e n ε ε e ε iε m e ε ε e ε iε m e ε t e ε i ε ε iε m e ε i ε e ε iε m ε iε ε iε ｉｓ ｉｎ ｉｎ ｉｎ 抗原? Immunol, 152: 193 (1994); Basak et al., “Application of the multiple antigenic peptides (MAP) state of the production of promoters: is, characterization and use of 8-branched immunogenic peptides "J.Pept.Sci, 1:. 385 (1995)). In a preferred method, multiple antigenic peptides are obtained from Tam, J. et al. P. "Multiple antigenic peptide system: A novel design for synthetic peptides and immunoassays", Synthetic Peptides: Approaches to Biologics. P. Tam and E.M. T. T. et al. Edited by Kaiser, Alan R. Liss, Inc. , New York, 3-18 (1989), by branching two peptides from a lysine residue.
[0075]
In one preferred embodiment, the MAP of the invention comprises at least 2, more preferably at least 3, and most preferably 4 copies of the peptide of the invention. A homogeneous MAP is a MAP that contains the same peptide in each of its arms. A heterologous MAP is a MAP that contains different peptides in its arms. In one embodiment, the homogeneous 4-arm MAP comprises SEQ ID NO: 5. In another embodiment, a homogeneous 4-arm MAP comprises SEQ ID NO: 6. In another embodiment, a homogeneous 4-arm MAP comprises SEQ ID NO: 7. In another embodiment, a homogeneous 4-arm MAP comprises SEQ ID NO: 8. In another embodiment, the homogeneous 4-arm MAP comprises SEQ ID NO: 9. In another embodiment, a homogeneous 4-arm MAP comprises SEQ ID NO: 10.
[0076]
In another embodiment, the MAP of the invention comprises a peptide having the sequence given by SEQ ID NO: 5 or an immunogenic fragment thereof, SEQ ID NO: 6 or an immunogenic fragment thereof, SEQ ID NO: 7 or an immunogenic fragment thereof, sequence No. 8 or an immunogenic fragment thereof, SEQ ID No. 9 or an immunogenic fragment thereof, SEQ ID No. 10 or an immunogenic fragment thereof, 10-25 residues in length, preferably 12-22 residues, or more preferably A 3-arm MAP having as an arm sequence any of the peptides of the present invention, comprising a fragment of SEQ ID NO: 2 that is about 15 residues. In one embodiment, the first arm of the 3-arm MAP comprises a peptide having the sequence given by SEQ ID NO: 5, the second arm of the 3-arm MAP has the sequence given by SEQ ID NO: 9, The third arm of the 3-arm MAP has the sequence given by SEQ ID NO: 10. In another embodiment of a three-arm MAP, the arms include SEQ ID NOs: 5, 6, and 7, respectively.
[0077]
As described above in the “Therapeutic Composition” section, the immunogenic amount of a peptide of the invention can be determined using standard procedures. For example, the initial immunization may comprise between about 1 μg and 10 mg, preferably between about 10 μg and 1 mg, and more preferably between about 50 μg and 500 μg of one or more peptides of the invention. Booster immunization is typically given to animals that receive the initial immunization. The general time conditions for booster administration are known in the art. In one embodiment, booster administration is given 3 weeks and 6 weeks after the initial administration. Booster doses typically contain about half the amount of peptide as the primary immunization.
[0078]
Standard techniques can be used to monitor the immunogenic response to immunity, as described in the “Therapeutic Composition” section above. For example, an immunological response can be determined using a nasopharyngeal (NP) challenge, as shown in the Examples section below. For NP challenge, a subject or animal, eg, a mouse, is treated with 10 Streptococcus pneumoniae suspended in 0.85% saline. ⁶ In colony forming units (cfu) can be challenged intranasally (IN). After sufficient time for bacterial growth (eg, 5 days after intranasal challenge), animals are sacrificed and nasal lavage performed, Wu, H. et al. Y. ("Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice" Microb. Pathog. 23: 127 (1997)). The wash can be diluted 3-fold to a final dilution of 1: 486. 50 microliters of each dilution can be cultured on blood agar + gentamicin plates (trypticase soy agar supplemented with 5% defibrinated sheep blood and 0.5% gentamicin). Data from NP colony formation and carrier in immunized mice and placebo (PBS) immunized controls can be analyzed using standard statistical tests such as t-test or Mann-Whitney rank sum test. Nasopharyngeal carrier is the number of colony forming units per nose. Nasopharyngeal colonization is either positive or negative for mice depending on whether at least 1 cfu is formed with 25 μl of nasal wash.
[0079]
The following examples are disclosed to provide those skilled in the art with a complete disclosure and description of the invention. These are intended as merely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[0080]
(Example)
(Bacterial strain) pneumoniae strain R36A is a strain of D. pneumoniae. E. Courtesy of Briles (University of Alabama at Birmingham). S. 24 serotypes pneumoniae is described in K. Provided by Facklam, Centers of Disease Control (CDC), Atlanta, Ga. These serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 11F, 12F, 14, 15B, 18C, 19A, 19F, 20, 22F, 23F and 33F. Enterococcus avium, E.I. cascriflavus and E. coli. Gallinarum is also described in R.A. Provided by Facklam. Anaerobic bacteria, R. Obtained from Dowell, CDC. These include Bacterioides asaccharolyticus, B.I. fragilis, B.M. intermedius, B.I. thetaiotamicron, Eubacterium lentum, Fusobacterium necrophorum, F. et al. nucleatum, Peptostreptococcus anaerobius, P. et al. Asaccharolyticus, Propionibacterium acnes, and Staphylococcus saccharolyticus. Branhamella catarrhalis and Bordetella parapertussis are described in R.A. Obtained from Weaver, CDC. Mycobacterium tuberculosis is described in R.A. C. Provided by Good, CDC. R. Barnes, CDC provided Chlamydia pneumoniae. The following remaining bacteria are derived from a stock collection of Immunology Laboratory, CDC: Bordetella pertussis, Enterobacter aerogenes, E. et al. agglomerans, E.M. cloacae, E .; gergoviae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae (types a to f), Legionella micdadei, L. et al. pneumophila, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Streptococcus agalactiae. equisimilis, S. et al. pyogenes and group G streptococci.
[0081]
(Production of MAb)
Female BALB / c mice were treated with S. cerevisiae, a crude derivative of capsular type 2 strain D39. Pneumoniae R36A was immunized with a whole cell suspension (Avery et al. (1994) J. Exp. Med. 79: 137-157). These mice were immunized by 3 intravenous injections and 1 intraperitoneal injection. The maximum number of cells injected at any given time is approximately 10 ⁶ Met. The fusion was performed on day 25 using standard procedures (Clafin et al. (1978) Curr Top, Microbiol. Immunol. 81: 107-109). Four mouse spleen cells were fused to Sp2 / 0-Ag14 myeloma cells (Schulman et al. (1978) Nature (London) 276: 269-270). The culture medium of the growing hybridoma is S. Antibodies against pneumoniae whole cells were tested by ELISA. The clone designated 1E7A3D7C2 was one of 10 selected for further study.
[0082]
(ELISA)
Screening of hybridoma culture supernatants was performed by ELISA. U-bottom microtiter plates (Costar, Cambridge, Mass.) Were diluted 1: 4,000 with 0.1 M carbonate buffer (pH 9.6). pneumoniae whole cell suspension (10 ⁹ cfu / ml) was sensitized with 50 μl and kept at 4 ° C. for 16 hours. The plate was washed 5 times with 0.9% NaCl (NaCl-T) containing 0.05% Tween-20. Culture supernatant (50 μl) from the fusion plate was added to 2% bovine serum albumin (BSA), 10% normal rabbit serum, 0.3% Tween in phosphate buffered saline (PBS) in the plate. -20, and 0.02% Merthiolate solution (pH 7.2) (ELISA dilution, Wells et al. (1987) J. Clin. Microbiol. 25: 516-521) and added to 50 μl and at 37 ° C. for 30 minutes Incubated. The plate was washed 5 times with NaCl-T. 50 μl of an ELISA dilution containing goat anti-mouse immunoglobulin horseradish peroxidase conjugate was added to each well. The plate was incubated at 37 ° C. for 30 minutes. The plate was washed and in 50 μl of 3,3 ′, 5,5′-tetramethylbenzidine (0.1 M sodium acetate with 0.005% hydrogen peroxide, 0.1 M citric acid, pH 5.7). 0.1 mg / ml) was added to each well and incubated at 37 ° C. for 30 minutes. The reaction is 4M H ₂ SO ₄ The optical density was read at 450 nm with a Dynatech ELISA Reader (Dynatech Laboratories, Inc., Alexandria, Va.). An optical density greater than 0.200 was considered positive.
[0083]
(SDS-PAGE and immunoblot analysis)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using an 8% acrylamide degrading gel by the method of Tsang et al. ((1983) Methods Enzymol., 92: 377-391). Cell suspension containing an equal volume of sample buffer (5% SDS-10% 2-mercaptoethanol-20% glycerol in 0.01 M Tris HCl, pH 8.0) and 2.4 μg protein per μl. The turbidity was mixed, heated at 100 ° C. for 5 minutes, and 5 μl of sample was applied to 1-15 wells. If the final protein content of the portion of the sample to be tested was less than 1.2 μg / μl, the sample volume up to 10 μl was applied to achieve a final protein concentration of 6 μl per well. Protein concentration was determined by the method of Markwell et al. ((1978), Anal. Biochem. 87: 206-210) using BSA as a standard. Proteins separated by SDS-PAGE were silver-stained by the method of Morrissey ((1981) Anal, Biochem. 117: 307-310) or electroblotted onto nitrocellulose (Schleicher & Schuell, Inc., Keene, N. .H.). This immunoblot procedure was performed according to a slightly modified method of Tsang (1983) et al. The blot was washed 3 times for 5 minutes with PBS (pH 7.2) containing 0.3% Tween-20 and gently stirred at 25 ° C. overnight (16 hours). The blot was blocked with casein-thimerosal buffer (CTB) for 1 hour (Kenna et al. (1985) J. Immunol Meth., 85: 409-419). After rinsing 3 times with CTB, the blot was exposed to goat anti-mouse immunoglobulin horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif) for 2 hours at 25 ° C. Conjugate dilutions (1: 2,000) were made with CTB. The blot was rinsed again 3 times with CTB and added to 3,3′-diaminobenzidine-4-hydrochloride (pH 7.2) (0.5 mg / ml) in PBS to 0.003% H ₂ O ₂ And 5 minutes exposure at 25 ° C. Reactivity was shown as a visible colored band on nitrocellulose paper. A low molecular weight protein standard (Bio-Rad) was used in PAGE and immunoblots. Rabbit antisera against protein standards were used to develop the standard (Carlone (1986) Anal. Biochem. 155: 89-91). Molecular weights were calculated by the method of Neville et al. ((1974), Methods Enzymol. 32: 92-102) using appropriate molecular weight standards.
[0084]
(Immunofluorescence assay)
A bacterial suspension (10 μl) containing approximately 400-500 cfu per field is placed on each well of an acetone resistant, 12 well (5 mm diameter) glass slide (25 × 75 mm) (Cel-Line Associates, Newfield, NJ). And dried at room temperature. The slide was then immersed in acetone for 10 minutes and allowed to dry at room temperature. MAb was added to the slide, which was incubated for 30 minutes at 37 ° C. Following incubation, the slides were gently rinsed with PBS and soaked twice at 5-minute intervals, blotted onto filter paper and allowed to air dry at room temperature. Null olescein labeled rabbit anti-mouse immunoglobulin (WF Bibb, Centers for Disease Control, Atlanta, GA) was then added and the slides were incubated at 37 ° C. for 30 minutes. They were then washed twice with PBS and gently blotted on filter paper. Slides are covered with carbonate buffered raised liquid (pH 9.0) and cover slip, then Leitz Dialux equipped with HBO-100 mercury incident light source, I stereoscopic filter system, 40 × dry objective and 6.3 × binoculars. Read using a 20 fluorescence microscope (Leitz, Inc., Rockeight, NJ).
[0085]
(Immunoelectron microscope)
Pneumococcal cells were washed twice with PBS and fixed in a freshly made mixture of 1% paraformaldehyde-0.1% glutaraldehyde at 4 ° C. for 20 minutes. The cells were dehydrated with a graded series of alcohols and then dehydrated for 1 hour at 4 ° C. in a 1: 1 mixture of complete ethanol and Lowicryl K4M (Ladd Research Industries, Inc., Burlington, VT). Cells were pelleted and suspended in a 1: 2 mixture of complete ethanol and Lowicryl K4M for 1 hour at 4 ° C. These were pelleted again and suspended in Lowicryl K4M (undiluted) at 4 ° C. for 16 hours. Cells were transferred twice to fresh and undiluted Lowicryl K4M during the next 24 hours. Lowicryl K4M treated cells were embedded in gelatin capsules and placed in an aluminum foil lined box. The capsules were cured using a short wavelength UV light source (72 hours at a distance of 35 cm, −20 ° C.). The box was brought to room temperature and the capsules were allowed to cure for up to 14 days. Capsule samples were cut into 100 μm thick sections and taken up on a nickel grid. The grid containing the sample was placed on a drop of ovalbumin solution in PBS containing sodium azide (EY Laboratories, Inc., San Mateo, Calif.) For 5 minutes. The lattice (wet) is transferred to a solution of primary MAb diluted in a solution of BSA reagent (0.1% Triton X-100, Tween 20, and 1% BSA in PBS containing sodium azide) (EY Laboratories). Transferred and incubated for 1 hour at room temperature or 18-48 hours at 4 ° C. in a humidified chamber. As an antibody binding control, another lattice was moistened with MAbs against Legionella pneumophila. The lattice was rinsed twice with PBS and incubated for 1 hour at room temperature on drops of colloidal cold particles (20 μm) (EY Laboratories) labeled with goat anti-mouse IgG. The grid was rinsed twice and post-stained with osmium tetroxide, uranyl acetate and lead citrate. The grid was tested using a Philips 410 transmission electron microscope.
[0086]
(CBA / CaHN / J mouse)
Wicker et al., Curr. Top. Microbiol. Immunol. 124: 86-101 X-linked immunodeficient (xid) CBA / N mice were used to study the protection conferred by the 37 kDa protein.
[0087]
(Example 1. Monoclonal antibody)
The MAb, Kohler et al. (1975 "Continuous cultures of fused cells secreting antibody of predefinded specificity", Nature 256: 495-497) produced by the method of, Zola et al. (1982, "Techniques for production and characterization of monoclonal hybridoma antibodies", J. G. Hurrell (eds.), Monoclonal hybridoma antigens: techniques and applications, CRC Press Inc., Boca Raton, FL, pages 1-57). The 37 kDa purified PsaA used for immunization of the mice pneumoniae serotype 22F and purified according to Tharpe et al. (1996, “Purification and serological activity of pneumococcal surface adhesin A (PsaA)”, Clin. Diagno. Lab Immunol. 29: 2 All MAbs were produced by immunization with PsaA purified from serotype 22F, except for 1E7 (1E7A3D7C2). pneumoniae R36A produced by immunization with a non-encapsulated strain (Russell et al., 1990, “Monoclonal antibody recognising a specific protein-specific protein from Streptococcus. PsaA was isolated using the procedures not shown in Examples 3 and 5 below. BALB / c mice were treated at 180 μg / ml in a 1: 1 emulsion with Freund's incomplete adjuvant (Sigma Chemical Co., St. Louis, MO) and phosphate buffered saline (pH 7.2). At the final concentration, the purified protein was first immunized intraperitoneally. One month later, mice were boosted with 110 μg / ml purified PsaA without adjuvant. Hybridoma fusions were performed using standard procedures (Clafin et al., 1978 "Mouse myelo-splen cell hybrids: enhanced hybridization procedures and rapid screening procedures", Curr. Topol. Spleen cells from two mice were fused with Sp2 / 0-Ag14 myeloma cells (Schulman et al., 1978, “A better cell line for hybridoma specific antigens”, Nature 276: 269-2). Sera from immunized mice and tissue culture supernatants from hybridized cells were subjected to routine blotting by ELISA using goat anti-mouse immunoglobulin-horseradish peroxidase conjugate and by standard Western blotting against standard PsaA. Were screened for reactivity to PsaA by SDS-PAGE in combination with Hybridomas that gave positive results in the screen were grown and used in peptide identification; these were 6F6 (6F6F9C8), 4E9 (4E9G9D3), 8G12 (8G12G11B10) and 1B6 (1B6E12H9). These MAbs were used in this study with 1E7.
[0088]
According to dot immunoblot and Western blot assays, these MAbs show 23 type-specific serotypes present in recent pneumococcal polysaccharide vaccines. Reacted with clinical isolates of Pneumoniae. Western blot confirmed that the detected antigen had a molecular weight of 37 kDa. S. In a developed study of 90 serotypes of pneumoniae, the 5 MAbs listed in the previous paragraph (not including 1E7) reacted with 89 of 90 serotypes (only 1B6 reacted with serotype 16F) could not). These listed MAbs are described in E. failed to react with E. coli, respiratory pathogens, or non-pathogens representing 22 genera and 29 species. MAb 1E7 reacted correspondingly with all pneumococcal strains tested (24 serotypes) and gave a sensitivity of 100%. For specificity, none of the 55 different bacterial non-pneumococcal strains (representing 19 genera and 36 species) reacted, thus obtaining 100% specificity.
[0089]
When required for use in the experiment described in Example 11, MAb was added to 0.1 M NaHCO with 100 μg N-hydroxysuccinimidyl-biotin ester (initially dissolved in DMSO). ₃ Biotinylation was performed by incubating 1 mg of protein in (pH 8.4).
[0090]
(Example 2. Cloning of Pneumococcal surface adhesion factor A gene)
Streptococcus pneumoniae DNA digested with the restriction enzyme Sau3A1 was ligated into BamHI digested pUC13 and E. coli. E. coli TB1 was transformed. Recombinant clones were identified by colony immunoblotting using a 37 kDa monoclonal antibody. Plasmid pSTR3-1 is an example of the pneumococcal surface adhesion factor A gene cloned into pUC13.
[0091]
Example 3. Preparation of purified PsaA
PsaA for use as an antigen or immunogen can be purified using any known method for protein purification. For example, Streptococcus pneumoniae can be routinely cultured and the cells harvested. The purified 37 kDa protein can then be isolated from the Streptococcus pneumoniae cell mass by extraction with a non-ionic detergent and can be further purified by ammonium sulfate fractionation and isoelectric focusing. (Tharpe et al., 1996, "Purification and serological activity of pneumococcal surface adhesin A (PsaA)" Clin. Diagnostic. Lab. Immunol. 3: 227-229).
[0092]
In another embodiment, an E. coli comprising an expression vector encoding a 37 kDa protein. The cells of the E. coli strain, TB1, can be cultured and harvested using conventional techniques. Examples of suitable expression vectors are vectors comprising a bacteriophage λPL promoter (eg, pKK1773-3), a hybrid trp-lac promoter (eg, pET-3a), or a bacteriophage T7 promoter. The 37 kDa protein (PsaA) can then be extracted from the cells using conventional methods for protein purification.
[0093]
(Protection experiment using 37 kDa protein)
Example 4
Twenty CBA / CaHN / J mice carrying the xid mutation (X-linked immunodeficiency) were used in this protection experiment. They were tested for protection against a challenge using the toxic capsular type 3 Streptococcus pneumoniae strain WU2. Mice were anesthetized with ketamine / rompan (Rompun) and blood was collected under the orbit to obtain pre-immune serum. A 37 kDa protein (pneumococcal surface adhesion factor (adhesin) A) was emulsified in complete Freund's adjuvant (CFA) at 54 μg per ml protein concentration. Ten mice were injected subcutaneously at 0.1 ml per site into two axillary sites and two inguinal sites, delivering approximately 22 μg protein / mouse. Ten control mice were treated independently with CFA and buffer instead of protein. After 14 days, 10 test mice were injected intraperitoneally (IP) with 100 μg of 37 kDa protein; controls were injected IP with buffer. Eight days after IP immunization, all mice were bled suborbitally to obtain post-immune serum and S. cerevisiae. Pneumoniae strain WU2 log phase culture 60 cfu was used to challenge intravenously (IV). Mice were observed for 21 days and deaths were recorded. Serum was collected before immunization to establish baseline exposure and was also collected after a complete immunization protocol (not pre-challenge) to correlate circulating antibodies against the 37 kDa protein with protection. The results obtained were as follows:
Days after challenge 1: No death
2: 3 control mice died
3: 2 control mice died
4: 2 control mice died, 1 control mouse was sick
5: 1 control mouse died
6-21: No dead mouse
Immunization with 37 kDa protein: 10/10 survival
Control without protein, 2/10 survival (8/10 death)
Different statistical significance: (p = 0.0008) rank sum test.
[0094]
(Example 5)
Twenty CBA / CaHN / J mice with xid mutations were injected according to the following protocol:
(1) All mice were bled before immunization to establish baseline immunity. Ten test mice were immunized subcutaneously at four sites with a total of 21 μg of 37 kDa protein antigen (PsaA) emulsified in complete Freund's adjuvant (CFA). Ten control mice were similarly immunized with CFA and buffer instead of antigen.
[0095]
(2) After 14 days, mice were booted with 100 μg of 37 kDa protein antigen (test mouse) or buffer (control) and intraperitoneally (IP). No adjuvant was used in this booster immunization.
[0096]
(3) After 8 days, all mice were bled via the orbital sinus and these were collected and pooled into two groups (immune and control). At the same time, blood was collected from individual mice and assayed for antibody response.
[0097]
(4) One day later, two additional mice were injected intraocularly with 0.1 ml pooled immune serum in an attempt to passively transfer immunity. Three additional mice were injected intraperitoneally (IP) with 0.1 ml pooled control mouse serum. (Because of the small amount of serum obtained from the immunized mice, only 5 mice were injected at this step).
[0098]
(5) One hour after IP injection, these five mice were treated with 140 colony-forming units (cfu) of S. cerevisiae in mid-log phase. Pneumoniae type 3 strain WU2 was used to challenge intravenously (iv).
[0099]
(6) At the same time, 18 (8 test, 10 control) mice were challenged iv with the same culture of WU2.
[0100]
(7) Mortality was counted daily.
Result: Number of deaths / total number of challenges
Immunization with a 37 kDa protein: 0/8
Control mouse: 10/10
Passive defense:
Mice receiving immune serum: 0/2
Mice receiving control serum: 3/3
Mice immunized with the 37 kDa protein were protected from fetal challenge with strain WU2; this immunity could be passively transferred using sera from the immunized mice. (Originally, 10 test mice were used, but 2 of these mice died of other causes before being immunized with WU2).
[0101]
(Example 6)
An enzyme-linked immunosorbent assay (ELISA) was used as a human antibody capture factor. Color was developed using purified 37 kDa protein antigen of Pneumoniae. Paired sera from children under 24 months of age known to have pneumococcal pneumonia were tested. Disease confirmation was determined by antigen in blood culture or urine. 35% (9/26) were found to have higher antibody titers than sera from non-sick children of the same age group (p = 0.06). This illustrates that some children responded to a 37 kDa protein antigen after natural infection.
[0102]
(Example 7)
(Preparation of 37 kD protein or polypeptide conjugate)
Conjugates can be prepared by use of a carrier protein that binds to a 37 kD protein or an immunogenic peptide or polypeptide derived from a 37 kD protein via a linker to elicit a T cell dependent response. The carrier protein can be any immunogenic protein such as, but not limited to, keyhole limpet hemocyanin, bovine serum albumin, tetanus toxin, diphtheria toxin, or bacterial outer membrane protein. Examples of bacterial outer membrane proteins useful as conjugates include Neisseria meningitidis and Haemophilus influenzae outer membrane proteins. Neisseria meningitidis can be selected from the group Neisseria meningitidis A, B or C. In addition, a 37 kD protein or polypeptide thereof can be used in the conjugate, where the 37 kD protein or polypeptide thereof is a T cell-dependent immunogenic carrier for a polysaccharide antigen that is a B cell stimulator. This is based on the theory that polysaccharide antigens are B cell stimulators and protective immunity is usually produced by a combination of B cell stimulators and T cell stimulants. The immune response induced by protein antigens exhibits T cell dependent properties (ie boosting and carrier priming). T cell dependent stimulators are important because many children under the age of 2 do not respond to T cell dependent antigens. Antigen adhesion or binding may be accomplished by conventional processes, such as those described in US Pat. No. 4,808,700. This includes additional chemicals that can form a covalent chemical bond between the carrier immunogen and the immunogen.
[0103]
The 37 kD protein antigens of the present invention can be administered to mammals, particularly humans, in a variety of ways. Exemplary methods include parenteral (subcutaneous) administration given with a non-toxic adjuvant, such as alum precipitate, or oral administration given after a decrease or disappearance of gastric activity or inactivation by gastric juice. Pharmaceutical forms that protect the antigen against (eg, protective capsules or microspheres). Dosage and dosing regimens mainly depend on whether the antigen is administered for therapeutic or prophylactic purposes, patient and patient history. The total pharmaceutically effective amount of antigen administered per dose typically ranges from about 2 μg to 50 μg per patient. For parenteral administration, the antigen is generally formulated in unit dose injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are essentially non-toxic and non-therapeutic. Examples of such vehicles include water, saline, Ringer solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as non-volatile animal oils and ethyl oleate can also be used. Liposomes can be used as vehicles. The vehicle may contain minor amounts of additives such as substances that increase isotonicity and chemical stability (eg, buffers and protective agents).
[0104]
(Example 8)
(Bacterial strain) All pneumoniae isolates were obtained from Streptococcal Reference Laboratories, Division of Bacterial and Mycologic Diseases, National Center for Infectious Diseases, Centers and Centers. The pneumococcal serotype 6B strain used for cloning and sequencing was the CDC reference strain (SP-86). E. E. coli DH5α (Bethesda Research Laboratories, Gaithersburg, MD) was used as the recipient host for the plasmid (pUC19 and its derivatives). S. The pneumoniae strain was grown on Trypticase soy agar plates with 5% sheep blood cells or grown on Todd-Hewitt broth with 0.5% yeast extract as shown here. E. E. coli cultures were grown in Luria broth. Where necessary, supplemented with 100 μg / ml ampicillin (Sigma Chemical Co., St Louis, Mo.).
[0105]
(Cloning and sequencing of the PsaA gene from S. pneumoniae, serotype 6B) A chromosomal library from pneumoniae serotype 6B was prepared as described above. (. Sampson et al., 1994, "Cloning and nucleotide sequence analysis of PsaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp.adhesins," Infect.Immun, 62:. 319-324) However, the pUC18 , Except that it was used as a cloning vector instead of pUC13. Recombinants were screened by colony immunoblot using monoclonal antibody 1E7 (Russell et al., 1990, “Monoclonal antibody recognition-specific protein-protein protein from Streptococcus pneumoniae. . This procedure and plasmid purification from positive clones (Ish-Horowicz et al., 1981, “Rapid and effective cosmid cloning,” Nucleic Acids Res. 9: 2898-2998) and restriction endonuclease analysis are all described previously (Sampson 1990, “Nucleotide sequence of httpB, the Legionella nepomophila gene encoding the 58-kilodalton (kDa) common antigen, formal deigned. 4). Dodecyl sulfate acrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed as before (Sampson et al., 1990). All other DNA manipulations were performed according to the method described in Sambrook et al. (Supra). DNA sequencing was performed using the ABI PRISM Dye Terminator Cycle sequencing kit and procedure (Perkin-Elmer, Cetus, Foster City, Calif). The DNASTAR software program (DNASTAR, Inc., Madison, Wis.) And the Wisconsin Genetics Computer Group sequence analysis software program (Fenno et al., 1989, “Nucleotide sequence of gene fifteen types of affiliation of the gene sequence.” .Immun.57: 3527-3533).
[0106]
(Preparation of genomic DNA for polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis) High molecular weight pneumococcal DNA was modified from Graves et al., 1993, “Universal DNA DNA isolation procedure,” pages 617-621. D. H. Pershing et al. (Ed.), Diagnostic molecular biology, (American Society for Microbiology, Washington, DC). Type-specific S. A 16 hour culture of Pneumoniae was grown in 50 ml of Todd-Hewitt broth containing 0.5% yeast extract in a screw cap flask without stirring at 37 ° C. Cultures were pelleted at 8000 × g for 15 minutes at room temperature and washed with phosphate buffered saline (10 mM, pH 7.2). The cell pellet was solubilized in 2.5 ml buffer consisting of 10 mM Tris, 10 mM EDTA (pH 8.0) and 0.4% SDS. 15 μl of protease K (20 μg / ml) was added and the lyase was incubated at 37 ° C. for 1 hour. The mixture was mixed by inversion and then adjusted to 0.48 M NaCl by addition of 500 μl 5 M NaCl and 400 μl 0.7% NaCl containing 10% hexadecyltrimethylammonium bromide was added. This suspension was mixed as before, incubated at 65 ° C. for 30 minutes and extracted with an equal volume of phenol-chloroform-isoamyl alcohol. The upper phase water was separated by centrifugation at 1500 × g and extracted with chloroform-isoamyl alcohol. DNA was precipitated from the upper aqueous phase over 30 minutes with 2.5 volumes of ethanol at -70 ° C. This was quantified by pelleting and drying in a desiccator, resuspending in water, and measuring the absorbance at 260 nm.
[0107]
(PCR-RELP) Restriction enzymes EcoRI, HinfI, MaeIII, MboII, MnII and NheI were obtained from Boehringer Mannheim Biochemicals (Indianapolis, Ind.); Purchased from. Primer sequences for the amplification reaction are described in S. pneumoniae serotype 6B gene (P1, AGGATCTAATGAAAAAATTTAG (SEQ ID NO: 3); P2.TCAGAGGCTTATTTTGCCAAT (SEQ ID NO: 4)) and the N-terminal sequence (nucleotides 181 to 201) and C-terminal sequence (nucleotides 1106-1126) of the adjacent region sequence Selected. This primer was synthesized using standard procedures.
[0108]
((I) DNA amplification) This reaction was performed using a Perkin-Elmer PCR amplification kit. Reaction volume is 100 μl and standard 1 × reaction buffer without Mg, 1 μM each primer, 2.0 mM MgCl ₂ 0.2 mM dNTP, template DNA and 2.5 UT Taq polymerase. The source of template DNA was either extracted purified chromosomal DNA or bacterial colonies. The conditions for amplification were as follows: 30 cycles of denaturation 94 ° C for 1 minute, annealing 52 ° C for 0.5 minutes and extension 72 ° C for 1.5 minutes. Amplified products were separated on a 1% agarose gel and visualized with ethylene bromide. A direct colony amplification procedure was employed. This shortens template preparation by removing the need for chromosomal DNA extraction. This procedure consisted of adding a single bacterial colony directly from the plate to the PCR mixture and heating at 95 ° C. for 10 minutes. The remaining PCR steps were outlined for extracted chromosomal DNA and performed as described above.
[0109]
((Ii) Enzymatic digestion) Digestion of the amplification product was performed as directed by the manufacturer for the specified enzyme in a volume of 20 μl. Digested products were analyzed by agarose (2% metaphor agarose, FMC Corp., Rockland, Me.) Gel electrophoresis and visualized after staining with ethylene bromide.
[0110]
(Analysis of type 6B PsaA) Genomic DNA was partially digested with Sau3AI, ligated into BamHI-digested pUC18 and E. coli DH5α was used to transform. Recombinant colonies are selected for ampicillin resistance and white colony formation in the presence of isopropyl-β-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indole-β-D-galactopyranoside did. Two positive colonies were obtained by colony immunoblot screening (using anti-PsaA Mab) of approximately 2,500 colonies. These were selected, purified and rescreened by Western blot analysis using the same MAb. Both of these expressed a protein reactive with the Mab against PsaA and migrated in SDS-PAGE with an expected molecular weight of approximately 37 kD. One was selected for continued studies and was named pSTR6. Limited restriction enzyme analysis of DNA from the recombinant plasmid showed that positive clones contained an insert that was 3.5 kb with sites for the enzymes ClaI, EcoRI and HindIII. In order to localize the PsaA coding region, the insert was double digested with SstI (multicloning site in the vector) and HindIII. This resulting fragment was ligated to pUC18 and E. coli DH5α was transformed. This produced a recombinant containing an insert of approximately 1.3 kb in size. The resulting subclone pSTR6y was shown to express full-length PsaA immunoreactive protein when analyzed by Western blot using SDS-PAGE and anti-PsaA Mab. The complete nucleotide sequence in both strands of the 1.3 kb insert was determined by circular sequencing of plasmid subclones using oligonucleotide primers complementary to this sequence. Sequence information was available. The nucleotide sequence of the complete streptococcal insert is shown in the sequence listing as SEQ ID NO: 1. A single open reading frame (ORF) begins at nucleotide 189 and ends at nucleotide 1,117, which encodes the PsaA gene sequence. This ORF is 930 nucleotides in length and is amplified and pGEM (Promega, Madison, Wis.) And BAC-to-BAC ^TM When subcloned into a vector system such as an expression system (Bethesda Research Laboratories, Gaithersburg, Md.), It expresses full-length PsaA reactive to anti-PsaA MAb antibodies. This ORF encodes a 309 amino acid peptide with an estimated molecular weight of 34,598 and an isoelectric point of 5.23. Analysis of peptides using the algorithm of Kyte et al., 1982 ("A simple method for displaying the hydrostatic character of a protein," J Mol. Biol. 157: 105-132.) Encoded the predicted leader sequence. It is shown to contain a major hydrophobic region of 20 amino acids. This leader contains a consensus sequence (LXXC) for signal peptidase cleavage. Removal of this leader yields a peptide of molecular weight 32,465 with an estimated isoelectric point of 4.97. Consensus sequence for ribosome binding site (Shine et al., 1974, “The 3′-terminal sequence of E. coli 16S ribosomal RNA: complementarity to nonsense triplets and ribos. 1324-1346) is located 5 nucleotides upstream of the ATG start codon.
[0111]
(Comparison of streptococcal homologue and serotype 6B sequence) Serotype 6B PsaA nucleotide sequence (Bilofsky et al., 1988, A GenBank gene sequence database, Nucleic Acids Res. 16: 1861-1864) (GenBank accession number U53509) and its adjacent and the region, stock R36A PsaA array has been previously published (Sampson et al., 1994, "Cloning and nucleotide sequence analysis of PsaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins Infect.Immun.62: 319-324) by comparison with the differences between the nucleotide sequences is shown. The calculated homology between the two sequences is 74%. The major regions of mismatch are the first 60 nucleotides that encode the upstream and downstream regions of the ORF and the putative signal peptide. When comparing two PsaA coding sequences, the sequence homology increases to 78%. The serotype 6B sequence was also compared to the PsaA DNA sequence for another vaccine serotype, serotype 2 (registration number U40786), recently submitted to GenBank. Computer analysis of these two sequences shows that they are similar. The calculated percent DNA homology between the two PsaA DNA sequences is 99%. There are 8 single base differences between the two sequences. Comparison of serotype 2 PsaA and serotype 6B PsaA shows almost complete identity: the calculated similarity value is 99.3%. The 8 base difference at the nucleotide level translates to a 6 amino acid peptide level difference with two changes resulting in conservative substitutions. Further analysis and viridans Streptococci and E. Comparison of the other 5 GenBank PsaA homologues from faecalis with serotype 6B sequences revealed significant sequence similarity (Fenno et al., 1989, “Nucleotide sequence of the type of fibrous gene band 13”). Infect.Immun.57: 3527-3533; Sampson et al., 1994. "Cloning and nucleotide sequence of PSAA, the Streptococcus pneumoniae gene encoding. . Ed Streptococcus sp adhesins, "Infect.Immun.62: 319-324:. Ganeshkumar et al., 1991," Nucleotide sequence of a gene coding for a saliva-binding protein (SsaB) from Streptococcus sanguis 12 and possible role of the protein in coaggregation with actinomyces. "Infect.Immun.59: 1093-1099, Kolenbrander et al., 1994." Nucleotide sequence of the Streptococcus gordonii PK488 coaggrega. Infect. Immun. s. et al., int., et al., et al., int., et al., et al., inc. 703-706).
[0112]
Sequence identity is 81% and 81% for PsaA (S. pneumoniae strain R36A), SsaB (S. sanguis), FimA (S. parasanguis), ScaA (S. gordonii) and EfaA (E. faecalis), respectively. 77%, 82% and 57%. Furthermore, all 6 sequences showed great similarity homology in the organism. Each contains a hydrophobic leader peptide that contains the prolipoprotein consensus sequence LXXC (for signal peptidase II cleavage) within the first 17-20 amino acids. It is clear that this N-terminal leader sequence represents the largest variable region. This is followed by a region of high similarity from amino acids 36 to 150. The region from amino acid position 150 to position 190 is a variable region, followed by another conserved region (amino acids 198-309).
[0113]
PCR-RFLP analysis of chromosomal DNA from 23 serotypes in 23-valent vaccine. PCR-RFLP was used to show 23 S. cerevisiae showing 23 serotypes in 23-valent vaccine. The degree of conservation of the PsaA gene between pneumonia serotypes was tested. Since previous attempts to amplify pneumococcal strains using primers corresponding to strain R36A were unsuccessful, PCR primers were used to determine the nucleotide sequence of the PsaA gene from serotype 6B, particularly the N-terminus of the PsaA polypeptide and Selection was based on the region of the gene encoding the C-terminus. S. Using a primer complementary to the PsaA gene from pneumoniae serotype 6B, the PsaA genes from all 23 serotypes and subtypes shown in 23 valent vaccines were amplified from chromosomal DNA. All 10 enzymes with restriction endonuclease digestion sites spanning the full length of the serotype 6B PsaA gene were selected. Nine out of ten enzymes produce the same pattern as all 23 PsaA genes analyzed.
[0114]
One exception, restriction enzyme Tsp509I, has 6 sites in the gene and produces 7 fragments that are digested to sizes of 7 bp, 30 bp, 68 bp, 146 bp, 151 bp, 166 bp and 362 bp. When these fragments are separated in a 2% metaphor agarose gel, five band patterns can be seen (7 bp and 30 bp fragments are not seen in these gels due to their small size). For 21 of the 23 serotypes, this five fragment enzyme pattern was seen; however, for the serotype 2 and 33F strains, the 146 bp fragment was absent and the two new fragments were 68 bp. Adjacent to this fragment, all resulting in 7 bands. Thus, the increase in the number of fragments results from the presence of an additional Tsp5O9I site within the 146 bp fragment.
[0115]
In order to confirm the incidence of this external site, the Tsp509I pattern of 3-4 additional strains of each of the 23 serotypes (no additional strains of serotype 2 and serotype 25 were available) analyzed. All the strains analyzed were random clinical isolates from the US provided to CDC for serotypes. The majority of the 80 strains are blood isolates; the exceptions were 2 strains from cerebrospinal fluid, 2 strains from pleural effusions, and 1 strain from the eyeball and nose, respectively. . 10% of the analyzed strains have an additional Tsp509I site that results in a modified RFLP pattern. This modification was only seen in types 4, 8, 11F and 33F. In an attempt to determine the incidence of this modified pattern, the PsaA gene from these four types of eight additional strains was analyzed for Tsp509I variations (all have ~ 11-12 for these four types) ).
[0116]
Table 1 summarizes the analysis of serotypes 4, 8, 11A and 33F. The altered pattern is sporadically present in serotypes 4 and 8, but is essentially always present in all 11A and 11F strains of 11A. Since strains that exhibit variation come from diverse regions within the country, this pattern of occurrence can be correlated to the geographic location or region of the United States. All strains of types 4, 8, 11A and 33F were blood isolates except for one 33F strain. 33F is a nasal isolate; therefore, the relevance of the site of isolation in the incidence of this modification cannot be assessed.
[0117]
Table 1. Screening of serotypes selected for additional Tsp509I restriction sites
[0118]
[Table 1]

^a Initial Tsp509I analysis including a survey of 2-3 strains of each of all 23 vaccine types
^b Tsp509I analysis of additional strains of the type exhibiting additional Tsp509I sites
^c Shown in parenthesis is the ratio of the number of serotypes with additional restriction sites to the number of serotypes tested.
[0119]
This analysis is described in S.H. Disclosed is the cloning and sequencing of the gene encoding PsaA from pneumoniae serotype 6B and the subsequent gene analysis in 23 pneumococcal polysaccharide vaccine serotypes. Sequence analysis revealed that the serotype 6B sequence and previously published strain R36A were not more similar than expected. The nucleotide sequence and its flanking regions were only 73% homologous to the originating strain R36A PsaA, whereas the actual PsaA coding sequence had a calculated homology of 78%. The protein sequence similarity between the two sequences was only 81%. By comparing the newly provided serotype 2 pneumococcal PsaA (vaccine serotype) and serotype 6B sequences, the DNA homology was calculated as 99% and 98% protein sequence similarity. Furthermore, these values are evidence that the sequence conservation for genes within the vaccine serotype is high. When comparing the deduced amino acid sequences of these two sequences to other published sequences for PsaA homology within the genus, regions of great similarity were evident in all five proteins. The similarity values within the group ranged from 57 to 82%.
[0120]
The need for Streptococcus pneumoniae vaccine candidates has been determined by the present inventors. The PsaA gene from pneumoniae serotype 6B was facilitated to be cloned and sequenced. Due to the heterogeneity between the two pneumococcal PsaA genes (6B and R36A), we tested vaccine serotypes and determined the degree of diversity between strains. S. Primers corresponding to the N-terminal and C-terminal coding regions of the PsaA gene from pneumoniae serotype 6B amplified all 23 vaccine serotypes. PCR-RFLP analysis using 10 different restriction enzymes representing 21 sites in the serotype 6B gene showed only one diversity region between strains, which is An additional Tsp509I site was created. This study demonstrates that the serotype 6B gene sequence is representative of sequences found among vaccine serotypes. Evidence for this includes 99% DNA sequence identity between serotype 2 and serotype 6B, and a uniform and identical restriction pattern covering the 21 sites tested in this study. . It is clear that the earlier strain R36A PsaA sequence represents a variant sequence that does not exist on the surface in the serotypes analyzed here. This is because the inventors were unable to amplify this sequence using primers against strain R36A PsaA. However, a more important aspect of this study is the limited diversity among the vaccine serotypes analyzed. It is the serotype that causes the disease and thus requires preventive measures against it. The lack of genetic diversity of PsaA among these serotypes suggests that the genes are highly conserved and are good candidates for vaccine development.
[0121]
(Example 9. Monoclonal antibody)
A 37 kDa protein from serotype 22F was used to generate monoclonal antibodies (1B6E12H9, 3C4D5C7, 4E9G9D3, 4H5C10F3, 6F6F9C8, and 8G12G11B10) (US Patent Application No. 08 / 715,131, now US Pat. No. 5,854). , 416, which are disclosed in the present specification by reference). These MAbs were analyzed for their ability to confer protection from infection by Streptococcus pneumoniae. Table 2 shows the capabilities of the five monoclonal antibodies tested, in particular one of the subsequent S. Efficient protection from the pneumoniae challenge was provided (8G12G11B10). S. Protection from Pneumoniae was dose responsive. This indicates that this monoclonal antibody was responsible for protection (Table 3).
[0122]
Table 2. Passive protection against Streptococcus pneumoniae serotype 6B conferred by anti-37 kDa monoclonal antibody in infant mice
[0123]
[Table 2]

^a Challenge dose (1.7 x 10 ³ cfu) or 10 × bacteremia dose 100% (BD ₁₀₀ ). Five mice per group received 50 μg of total antibody. All MAbs are IgG.
[0124]
Table 3. Effect of the second dose on the potential passive protective capacity of the anti-37 kDa monoclonal antibody 8G12G11B10
[0125]
[Table 3]

^a All infant mice are 10 × BD ₁₀₀ (2 × 10 ³ cfu) challenged. MAbs were given 24 hours before challenge (before) and 24 hours after challenge (after). 10 mice / group.
[0126]
(Example 10. Phage display library)
A phage display library containing an insert of 15 amino acid residues located at the N-terminal portion of the pIII coating protein (Parmly and Smith, 1988) was constructed in phage FUSE 5 as a vector. This library was ligated with a 33 bp synthetic BgII fragment to FUSE 5 and E. coli. E. coli Kqll / kan + cells were generated by transfection by electroporation. This phage progeny contains the display library.
[0127]
(Example 11. Screening by biopanning)
Four cycles of biopanning were performed for each MAb used for phage with the aim of screening the phage display library of Example 10 for PsaA epitope peptides (Smith and Scott, 1993, Meth. Enzymol., 217: 228- 257). The Petri dish substrate was coated with streptavidin and 10 μg of biotinylated anti-PsaA MAb as prepared in Example 1. The remaining biotin binding sites were blocked with 1.5 ml D-biotin (10 mM). This phage library (10 ¹¹ -10 ¹² Transformants) were incubated with immobilized MAbs. Bound phage was eluted from the streptavidin-coated plate with 0.1 N HCl, pH 2.2. The eluted phage was titrated and amplified and then subjected to two additional selections performed as described above. The amount of biotinylated MAb used was 1 nM and 1 pM in the second and third rounds, respectively, so that only the high affinity peptides were bound at the end of the last cycle.
[0128]
Example 12 Amino Acid Sequence of Immunogenic Peptide
The library-derived high affinity peptides obtained using the procedure of Example 11 were grown and sequenced. For each MAb, 10 phage specimens resulting from the selection process were sequenced. About 1 μg of single stranded DNA was purified by phenol-chloroform extraction, ethanol precipitated and resuspended in 7 μl of water. A 27-mer primer complementary to the FUSE 5 vector sequence derived from the region in wild-type pIII, common to all fd-tet derived vectors, and ³⁵ Sequencing reactions were performed using S Sequenase version 2 (US Biochemicals, Cleveland, OH). The resulting sequences are shown in Table 4. These were compared to the known sequences of PsaA strains 2 and 6B using the ClustaIV and tFasta programs to identify the epitope for PsaA where each peptide was closest aligned. These epitope positions are also shown in Table 4. Peptides obtained using MAbs (8G12, 6F6 and 1E7) if additional residues are present in this protein, where a gap appears after residue 13 of this peptide (SEQ ID NO: 7 and SEQ ID NO: 8) Aligns best with PsaA.
[0129]
(Table 4. Peptide sequences obtained by biopanning using MAb)
[0130]
[Table 4]

Example 13. Immunization of mice with an immunogenic peptide of PsaA
Peptides having the sequences shown in SEQ ID NOs: 5, 6, 7, and 8 can be synthesized by conventional methods (eg, using an automated peptide synthesizer). The peptide can be purified by reverse phase HPLC and the major peak can be collected. Peptide sequences can be confirmed by automated peptide sequencing using an automated sequencing device (eg, an automated sequencing device manufactured by Beckman Instruments, Inc., Mountain View, Calif.). Each peptide can be coupled to an adjuvant such as keyhole limpet hemocyanin using coupling mediated by a water soluble carbodiimide reagent. The resulting conjugate can be dissolved in phosphate buffered saline pH 7.2 to a final concentration of about 180 μg / ml and in a Freund's incomplete adjuvant (about 1: 1 ratio) in the emulsion. Sigma Chemical Co., St Louis, MO). BALB / c mice can be first immunized intraperitoneally with this suspension, and one month later the mice can be boosted with about 110 μg / ml conjugate without adjuvant.
[0131]
Example 14. Immunization of mice with consensus peptides identified by phage display
A peptide with a consensus sequence (SEQ ID NO: 8) from the sequence of several peptides identified by phage display was tested for its ability to elicit an immunological response to PsaA. This consensus peptide (LVRRFVHRPRPHVESQ; SEQ ID NO: 7 derived from a peptide that binds to monoclonal antibodies 8G12, 6F6 and 1E7 in the phage display experiment described in Example 12 above ) Was synthesized with a CYGG spacer and a lauroyl group at its amino terminus (Bio-Synthesis, Lewisville, TX). This lauroyl group enhances the hydrophobic complex formation of peptide groups to the proteosome (Lowell, GH, et al., “Proteome, hydrophobic anchors, iscoms, and liposomes for imprinted presentation in N.). , Woodrow, GM, Levine, MM (eds.), Marcel Dekker, Inc., New York, pages 141-160 (1990); Lowell, GH, et al., “Peptides bound to proteosomes biosciences. fee become high immunology ic withoutadjuvants "J. Exp. Med. 167: 658 (1988); and Zollinger, WD, et al.," Complex of meningococcal group B polysacandide and type 2 mm. : 836 (1979)). On the other hand, this cysteine group enhances the immunogenicity of the peptide (Lowell, GH et al. (1990)). As described by Zollinger (Zollinger et al. (1990)), proteosomes were isolated from group B. Prepared from outer membrane complex vesicles from meningococci, strain 99M. Synthetic lipopeptides were complexed to the proteosome in a 1: 1 (w / w) ratio by combining the components in the presence of a surfactant. This surfactant was removed by extensive dialysis (Lowell, GH et al. (1988)).
[0132]
3-4 week old BALB / c mice (n = 4 per group) were primed at week 0 with 10, 25, or 50 μg peptide (SEQ ID NO: 5) / proteosome complex. The mice in each group were then boosted at 10, 25, or 50 μg peptide (SEQ ID NO: 5) / proteosome complex at 2 and 3 weeks. Mice primed with 20 μg PsaA protein at week 0 were positive controls for this study. Negative controls consisted of mice primed with 10, 25, or 50 μg proteosome at week 0 and boosted with 10, 25, or 50 μg proteosome at weeks 2 and 3. Serum was collected weekly for 4 weeks and anti-PsaA specific antibodies were tested by enzyme-linked immunosorbent assay (ELISA).
[0133]
An anti-PsaA specific ELISA was performed as follows: Nunc immuno Maxi Sorb plates were coated overnight at 4 ° C. with 5 μg / ml of purified native PsaA protein. Plates were washed with PBS / Tween buffer (PBS containing 0.01% Tween-20) and blocked with PBS / Tween buffer containing 1% BSA. Serial dilutions of mouse serum starting at 1:10 in PBS / Tween / BSA were incubated for 1 hour at 37 ° C. The plate was washed 4 times with PBS / Tween. Anti-mouse IgG and IgM (Sigma, St. Louis, MO) conjugated to horseradish peroxidase, diluted 4000: 1 in PBS / Tween / BSA, were added to the plates. Anti-PsaA antibody was detected in the dark using an o-phenylenediamine substrate for 30 minutes. Absorbance was read at 490 nm with Microplate E1311 (Biotek, Winooski, VT).
[0134]
All sequences of peptides selected by MAbs 8G12, 1E7 and 6F6 were aligned to the same region of the PsaA molecule. One peptide selected by MAb 8G12 had the same sequence as the peptide selected by 1E7 and 6F6 (SEQ ID NO: 8) as shown above. The homology between these peptides and PsaA was determined and was present at 6 amino acids. This region was used to define the consensus peptide sequence (SEQ ID NO: 8). This peptide was synthesized with an N-terminal lauroyl group and a CYGG motif, complexed to the proteosome, and improved immunogenicity. Mice were immunized with 10, 25, or 50 μg of consensus peptide complexed to the proteosome. Sera from peptide immunized mice showed an anti-PsaA response. The results of this study are shown in Tables 5 and 6. The peptide immunized mice developed an IgG response, with titers ranging from 159 to 252 at 4 weeks. This titer was calculated after normalizing the data with respect to proteosome background values. Of the three dose groups, the immune response to the 50 μg peptide dose group was the highest. However, the anti-PsaA response to this peptide was significantly lower than the immune response to PsaA protein (p <0.05).
[0135]
(Table 5. Potency of IgM against PsaA)
[0136]
[Table 5]

This data is presented as a 25% titer of control serum from mice immunized with 20 μg PsaA.
[0137]
(Table 6. IgG titer against PsaA)
[0138]
[Table 6]

As in Table 5, this data is presented as a 25% titer of control sera from mice immunized with 20 μg PsaA.
[0139]
Example 15. Immunity of mice with fat- and non-lipid-soluble forms of peptides identified by phage display (data from Srivastava, N. et al., Hybridoma 19:23, 2000)
A peptide having the amino acid sequence of SEQ ID NO: 5 (T V S R V P W T A W A F H G Y) and selected by phage display using MAb 4E9 was obtained from S. cerevisiae. We tested for its ability to protect mice from pneumoniae challenge. Two forms of this peptide were synthesized: one with a palmitoyl residue at its N-terminus (“SEQ ID NO: 5-lipidated”) and the other with such derivatization. None (“SEQ ID NO: 5-unlipidated”). These peptides are described in Stewart et al., “Solid peptide synthesis”, 2nd edition, Pierce Chemical Co. , Rockford, IL (1984), using solid phase F-moc chemistry and synthesized using an ACT model 396 HPS peptide synthesizer (Advanced ChemTech, Louisville, KY). Lipid solubilized versions of the peptides described above containing monopalmitic acid, palmitic acid (Sigma Chemicals, St. Louis, Mo.), and standard amino acid linkage described above (Verhaul et al. (1995)). Synthesized by attaching to the deprotected amino terminus of the resin-bound peptide using the same reaction conditions. The sequence was verified by automated peptide sequencing using a Porton model # 2090 automated peptide sequencer (Beckman Instruments, Inc., Mountain View, CA). The peptide was suspended in phosphate buffered saline pH 7.2 (PBS) to a final concentration of 1.0 mg / ml for the first dose and 0.5 mg / ml for subsequent doses.
[0140]
Peptide SEQ ID NO: 5-lipid solubilized and SEQ ID NO: 5-non-lipid solubilized To analyze the ability to protect against pneumoniae challenge, 10-week-old ND-4 mice (Swiss Webster) were immunized using a 3-dose regimen. Test mice (n = 15 for each peptide) received an initial dose of 100 μg peptide on day 0, followed by a booster dose of 50 μg peptide at weeks 3 and 5. This peptide SEQ ID NO: 5-lipid solubilized was suspended in 100 μl PBS 0.01M, pH 7.2, while the non-lipid solubilized peptide (SEQ ID NO: 5-non-lipid solubilized) was added to the adjuvant alhydrogel (2%; # A1090BS, Accurate Chemical and Scientific Company, Westbury, NY) was mixed at a concentration of 6.3 mg / ml in PBS to enhance the immunogenicity of this peptide. Control mice (n = 12) were similarly immunized except that no peptide was used. Each mouse was immunized subcutaneously between the shoulders. One week after the final boost, all mice were 4.9 x 10 ⁶ cfu S. pneumoniae strain PLN-D39 (contributed by James Paton, Women's and Children's Hospital, North Adelaide, SA Australia), which is a pneumolysin negative derivative of D39. . Five days after this, they were euthanized and cultured with nasal wash. PBS nasal washes were performed according to Wu, H. et al. Y. ("Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice", Microb. Pathog. 23: 127 (1997)). Each wash was diluted 3-fold to a final dilution of 1: 486. 50 μl of each dilution was cultured on blood agar + gentamicin plates (Trypticase soy agar supplemented with 5% defibrillated sheep blood and 0.5% gentamicin). Data from NP colonization and carriage in immunized mice and placebo (PBS immunized control) were analyzed using either the t-test or the Mann-Whitney rank sum test. Nasopharyngeal carrier is the number of colony forming units per nose. Nasopharyngeal colonization is either positive or negative for mice, depending on whether at least 1 cfu is formed in 25 μl of nasal wash.
[0141]
S. Peptide SEQ ID NO: 5-lipid solubilized and SEQ ID NO: 5-non-lipid solubilized. To analyze the ability to protect against the pneumoniae challenge, Swiss Webster mice were immunized as described above with SEQ ID NO: 5-lipid solubilized and SEQ ID NO: 5-non-lipid solubilized, and PsaA. Mice immunized with peptide SEQ ID NO: 5-lipid solubilized showed a statistically significant reduction (p <0.05) in bacterial nasal retention when compared to placebo controls (Table 7). Furthermore, the peptide immunized with peptide SEQ ID NO: 5 non-lipid solubilized The chance of being resistant to colonization by Pneumoniae serotype 2 was 40% higher (Table 7). This was statistically significant when compared to placebo control (p <0.05, Fisher's direct test, two-sided test). A decrease in pneumococcal nasal colonization was found in mice immunized with non-lipid solubilized peptide SEQ ID NO: 5-non-lipid solubilized; however, this decrease was not statistically significant when compared to placebo controls. (P = 0.48) (Table 7).
[0142]
(Table 7. Summary of protection studies in Swiss-Webster mice. ^* )
[0143]
[Table 7]

^* This data is presented as the geometric mean of the three experimental carriers (cfu / nose). Colony formation is defined as 1 cfu / 25 μl nasal wash. This significance is calculated using the Mann-Whitney U rank sum test (p <0.05).
^** Fat solubilization with monopalmitic acid at the N-terminal additional cysteine residue. The lipid solubilized peptide contained the amino acid sequence CSS linked to the amino terminus of the amino acid sequence from the sequence listing shown so that the cysteine (C) residue is at the amino terminus of the peptide.
[0144]
Example 16 Immunization of mice with multiple antigenic PsaA peptides
S. of peptides having the sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. The ability to protect mice from pneumoniae challenge was examined when administered separately in the lipid solubilized or multi-antigenic peptide (MAP) form or when combined with MAP. Peptides having the sequences given in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 were synthesized using similar reagents, equipment, procedures as described in Example 15. Multiple antigenic peptides (MAPs) containing either two arms, three arms, or four arms (FIGS. 1A-1C) were obtained from Tam, J. et al. P. , "Multiple antigenic peptide system: A novel design for synthetic peptides and immunoassays" Synthetic Peptides Approaches Approaches Approaches. P. Tam and E.M. T. T. et al. Edited by Kaiser, Alan R .; Liss, Inc. , New York, 3-18 (1989). All MAPs described in this example contain an additional norleucine (NLe) residue at the carboxy terminus to facilitate synthesis (FIGS. 1A-1C). Alum was added to this suspension for non-lipid solubilized peptides.
[0145]
Separate groups of ND-4 mice (n = 8 per peptide group and control group) were treated with 100 μg, 50 μg, or 50 μg of non-fat solubilized peptide or fat three times, week 0, week 3 and week 5, respectively. Immunized subcutaneously with either of the solubilized peptides. One week after the third dose, mice were suspended in 10 μl of 0.85% saline, 10 of Streptococcus pneumoniae serotype 2, serotype 4, or serotype 6B. ⁶ Attacked intranasally (IN) with cfu. Five days later, they were euthanized and the nasal washes were cultured. Nasopharyngeal (NP) colonization and carriage were analyzed as described in Example 15.
[0146]
Immunization with non-lipid solubilized or lipid solubilized monophasic peptide MAP (ie homogeneous) and biphasic peptide MAP (ie heterogeneous MAP containing two different peptide sequences) provides in vivo protection against intranasal carriage (Ie, a decrease in cfu), such that S. Produces an immunological response to pneumoniae challenge. The strongest immunological response was observed with immunization with the biphasic peptide MAP. A decrease in NP colony formation was observed with lipid solubilized and non-lipid solubilized peptides and various MAP constructs, and this decrease varied between them. Immunization with non-lipid solubilized monophasic peptide (ie, homogeneous) MAP reduced NP colony formation between 40% and 93% (Table 8). Immunization with fat-solubilized monophasic peptides reduced NP colony formation between 48% and 94%. The greatest reduction in carriage was observed with the biphasic peptide MAP. Immunization with non-lipid solubilized biphasic peptide MAP reduced NP colony formation between 68% and 91% (Table 9). This reduction in NP colony formation observed with immunization with the biphasic peptide MAP was statistically significant (p <0.05) for all serotypes and biphasic peptides tested.
[0147]
The PsaA peptide used in the experiment, identified by phage display analysis using monoclonals against PsaA, is immunogenic and this is the same as the S. cerevisiae tested. The carriage of three serotypes of pneumoniae was reduced. Furthermore, the biphasic peptide MAP was more efficient in reducing carriage than the monophasic peptide MAP.
[0148]
(Table 8. Decrease in nasopharyngeal (NP) colony formation (%): non-lipid solubilized 4-arm homogeneous MAP compared to control (P value)
[0149]
[Table 8]

^* Non-lipid solubilized peptide (NL).
^** Statistical significance (P ≦ 0.05) from the control was calculated by t-test or Mann-Whitney rank sum test.
^*** Homogeneous 4-arm MAP of SEQ ID NO: 5.
^*** Homogeneous 4-arm MAP of SEQ ID NO: 6.
^{＊＊＊＊＊} Homogeneous 4-arm MAP of SEQ ID NO: 7.
[0150]
(Table 9. Reduction in NP colony formation (%) of fat-solubilized peptide MAP of the present invention and biphasic peptide MAP compared to control) ^* )
[0151]
[Table 9]

^* (P value).
^** Fat-solubilized with monopalmitic acid at the additional cysteine residue at the N-terminus. The fat solubilized peptide includes the amino acid sequence CSS that binds to the amino terminus of the amino acid sequence from the sequence listing shown so that the cysteine (C) residue is the amino terminus of the peptide.
^*** Heterogeneous 4-arm MAP of SEQ ID NO: 5 and SEQ ID NO: 6 as shown in FIG. 1A.
^*** Heterogeneous 4-arm MAP of SEQ ID NO: 5 and SEQ ID NO: 9, as shown in FIG. 1B.
[0152]
Example 17. Immunization of mice with the triphasic peptide MAP
Homogeneous (monophasic peptide) 3-arm MAP and heterogeneous (biphasic or triphasic peptide) 3-arm MAP are It can be used to provide protection against pneumoniae attacks. For example, as described above for monophasic peptide MAP and biphasic peptide MAP, a triphasic peptide 3-arm MAP or an immunogenic fragment thereof comprising any combination of SEQ ID NO: 5 to SEQ ID NO: 10 (eg, A homogeneous triphasic peptide MAP comprising 3 arms, each having the amino acid sequence shown in SEQ ID NO: 5, or a first arm having the sequence of SEQ ID NO: 5, a second arm having the sequence of SEQ ID NO: 9, and a sequence Heterogeneous triphasic peptide MAP comprising a third arm having the sequence of number 10 (see, eg, FIG. 1C) for similar reagents, devices, and as described in Examples 15 and 16 The procedure can be used to synthesize and test. Other such examples of triphasic peptide combinations include, but are not limited to: a first arm comprising SEQ ID NO: 5, a second arm comprising SEQ ID NO: 6, and SEQ ID NO: 7, sequence A third arm comprising SEQ ID NO: 5, a first arm comprising SEQ ID NO: 5, a second arm comprising SEQ ID NO: 10, and a third arm comprising SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 Three arms; a first arm comprising SEQ ID NO: 6, a second arm comprising SEQ ID NO: 7, a third arm comprising SEQ ID NO: 9 or SEQ ID NO: 10, and the like.
[0153]
Example 18. Inhibition of pneumococcal carriage in mice by subcutaneous immunization with peptides derived from PsaA
We have identified the ability of MAP to inhibit the pneumococcal (Pnc) nasopharyngeal (NP) carriage in immunized mice, either in lipophilic form or in combination with each other (two biphasic peptides). And three MAPs) were tested. Inhibition of the NP carriage was dramatically increased compared to homogeneous MAP alone using a combination of MAP and a biphasic peptide. These observations indicated that this anti-PsaA MAb phage display expressed peptide is immunogenic and significantly reduces NP carriage in mice. These PsaA peptides are potentially useful as vaccines for humans and / or other animals for the reduction and / or elimination of Pnc carriage, otitis media, and / or invasive Pnc disease.
[0154]
(A) Material and method)
(Bacterial isolates and growth conditions)
S. Three isolates of pneumoniae (representing serotype 2, serotype 4 and serotype 6B) were used for NP carriage experiments. The serotype 2 isolate PLN-D39 was kindly provided by James Paton (Women's and Children's Hospital, North Adelaide, SA, Australia); and the serotype 4 isolation Body DS2341-94, and isolate DS 1756-94, which is 6B, were kindly provided by Richard Facklam (Centers for Disease Control and Prevention, Atlanta, GA). These selected isolates are representative strains and were frequently used in animal model experiments in our laboratory and elsewhere. To ensure that multiple experiments could be initiated from the same lot of cells, a standardized stock culture of each pneumococcal isolate was prepared as described above (Johnson SE et al., J. Infect. Dis. 180: 133-40, 1999). At the start of the experiment, stock cells were cultured on blood agar medium (BA, Trypticase soy agar supplemented with 5% defibrinated sheep blood; BBL, Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) Moved to brain heart infusion broth (BHI; BBL) supplemented with 10% Levinthal basal medium (BHI-10L; BBL) and engineered for animal inoculation as previously described (Johnson SE Supra).
[0155]
(animal)
Adult Swiss Webster mice (ND-4; female; 8 weeks old) were obtained from Harlan Sprague Dawley, Inc. , Indianapolis, IN. Upon arrival, mice were placed in groups of 8 per cage. All animals were housed under standard conditions (25 ° C., about 40% relative humidity) with any food and water intake. All mice were allowed to adapt to these new environments one week prior to the experiment.
[0156]
(Peptide synthesis)
When the amino acid sequences of the aforementioned peptides (base peptides denoted P1, P2 and P3) are used as templates, they are both fat-solubilized and non-lipid-solubilized forms and are homogeneous in three, 4-arms Multiple antigenic peptides (MAP) were synthesized with non-lipid solubilized biphasic (4-arm heterogeneous) peptide constructs and triphasic peptide (3-arm homogenous) constructs and studied in a mouse NP carriage model. These MAPs have been described previously (Barany, G and Merrifield, RB, The Peptides, Vol. 1, Gross, E and Meienhofer, J, Ed., New York: Academic Press, 1980, pages l-284; Verheul. ACT 396, using standard and modified Fmoc protocols, such as J. Immunol. Meth. 182: 219-226, 1985; and Reed, RC, et al., Vaccine 15: 482-488, 1997). Synthesized with a multiple peptide synthesizer (Advanced ChemTech, Louisville, KY). The biphasic and triphasic peptide constructs were obtained from Tam's method (Tam JP, Multiple antigenic peptide system: A novel design of synthetic chemistry, Al., Et al., Et al. , Inc., 1989, pp. 3-18) using Fmoc-Lys (Dde) or Fmoc-Lys (ivDde) (Novabiochem, San Diego, Calif.) With piperidine and hydrazine. synthesized by differential detection). This crude peptide is subjected to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry followed by one or more procedures of amino acid analysis, high performance liquid chromatography (HPLC), capillary electrophoresis and / or peptide sequencing. , Analyzed for synthesis fidelity. If necessary, the peptide was purified by semi-preparative HPLC using an acetonitrile / water / trifluoroacetic acid buffer system.
[0157]
Structurally, this MAP is homologous to each other and contains four branches consisting of either P1, P2 or P3. The fat-solubilized form (shown as P43, P44, and P45) and the non-fat-solubilized form (shown as P46, P47, and P48) are those in which the former is a three amino acid linker (cysteine- It is structurally identical except that it has a monosubstituted palmitoyl residue linked by (serine-serine) (FIGS. 2A and 2B). The MAP was solubilized with palmitic acid to study the effect of adjuvants on peptide immunogenicity and subsequent effects on the NP carriage. The biphasic peptides termed P79 and P80 are a combination of base peptides (P1 and P2, or P1 and P3) (FIG. 3). P79 contains two units of each of P1 and P2, while in P80 P2 is replaced with P3. The triphasic peptide (identified as P81 and consists of three branches) is composed of one unit of each of the base peptides (Figure 4). In all cases, this peptide branch was synthesized based on either a 3-arm leucine-norleucine core or a 4-arm leucine-norleucine core (FIGS. 1-3).
[0158]
(Mouse immunization and challenge)
The mouse NP carriage model (described above) was used to determine the effect of immunogenic PsaA peptide on inhibition of the NP Pnc carriage (Lipschitch, M et al., Vaccine 18: 2895-901, 2000). Mice were immunized based on a 3 dose regimen. Test mice (n = 8 for each peptide or peptide construct) received a starting dose of 100 μg on day 0, followed by a 50 μg booster dose of the appropriate peptide 3 and 5 weeks after the first dose. It was. This fat-solubilized MAP was resuspended in 100 μL sterile 0.01 M, pH 7.2, phosphate buffered saline (PBS), but all other peptides were resuspended in 100 μL PBS. Mixing with 3 mg / mL adjuvant alum (alhydrogel-2%, # A1090B, Accurate Chemical and Scientific Company, Westbury, NY) to increase the immunogenicity of this peptide. Control mice (n = 8) were immunized in the same manner, but no peptide was used. Each mouse was immunized subcutaneously between the shoulders. One week after the final boost, all mice were injected intranasally with the appropriate S. coli suspended in 10 μL of 0.9% sodium chloride buffer (SCB; Abbott Laboratories, North Chicago, IL). 1-3 × 10 of pneumoniae isolate ⁶ Attacked with colony forming units (cfu). Challenge inoculum was serially diluted 10-fold and plated on BA plates to determine the challenge dose.
[0159]
(Nasal irrigation and culture)
On day 5 post challenge, each mouse was euthanized and the nasopharyngeal cavity was emptied with 100 μL SCB as described by Wu et al. (Wu, HY et al., Microb. Pathog. 23: 127-37, 1997). Washed. This wash was serially diluted 3 times to a final dilution of 1: 486. 50 μl of each dilution was cultured on BA + gentamicin plates (BA plates supplemented with 0.5% gentamicin). The culture is 5% CO ₂ Incubated overnight at 37 ° C. in an atmosphere and high humidity. When possible, clones were counted and recorded for each dilution. The carriage for a particular mouse was defined as the number of cfu in 50 μL of collected nasal washes after each dilution count was adjusted to the undiluted level and averaged. The detection sensitivity of the carriage assay is 40 cfu / (ml nasal wash).
[0160]
(Statistical method)
Inhibition of the Pnc carriage was determined by calculating significant differences in the NP carriage between immunized test mice and control mice without these paired immunogens. Significant differences between groups were calculated by Mann-Whitney rank sum test. The risk factor was set at P <0.05. Statistical calculations were performed using SigmaStat software, version 2.0 (Jandel Scientific Co., San Rafael, Calif.). The analysis is based on data generated from a single test for each of the Pnc serotype and immunogen formulation.
[0161]
(B) Result)
(MAP effect in NP carriage inhibition)
In preliminary experiments, an immunological response to MAP P43 was observed in mice when the mice were immunized according to the schedule as described above (data not shown) (titers were consistently greater than 1: 5,200). Met). Based on these results, Pnc carriage inhibition experiments were initiated with the described peptides in adult mice. Mice were immunized with non-lipid solubilized MAP, P46, P47, or P48. When challenged with S. pneumoniae serotype 2, serotype 4, or serotype 6B, significant (P <0.05) carriage inhibition was compared to controls and in median number of cfu colonizing the NP cavity. An observation was made when there was a reduction of more than 60% for the carriage when specified (Table 10). P46 significantly reduced serotype 2 and serotype 4 NP colony formation (P <0.05), but not serotype 6B colony formation (P = 0.11). P47 and P48 significantly (P <0.05) each reduced colony formation of one serotype, ie serotype 4 and serotype 6B, respectively. Similar results were observed when mice were immunized with fat solubilized MAP P43, P44, and P45 (Table 11). When Pnc colonization was reduced by 68%, nasopharyngeal carriage inhibition was significant (P <0.05). Serotype 4 carriage was significantly (P <0.05) inhibited by each of these MAPs and serotype 6B, serotypes P43, and P44. However, these MAPs did not demonstrate significant (P <0.05) carriage inhibition against serotype 2 but reduced 70% in serotype 2 NP colony formation in mice immunized with P45 For these significantly (P <0.05) inhibited serotypes, the average number of Pnc bacteria colonizing the NP cavity is non-lipid solubilized and fat solubilized. For MAP, it was reduced to 58% and 71%, respectively.
[0162]
(Effects of MAP combinations or polypeptides on NP carriage inhibition)
Given the promising results for MAP carriage inhibition by MAP, further studies were carried out using MAP combinations and polypeptide constructs. In all cases where mice were immunized with MAP combinations (P43 + P44 or P43 + P44 + P45) or biphasic peptides (P79 or P80), significant (P <0.05) inhibition of the NP carriage was observed (Table 11). The level of NP colony formation was significantly (P <0.05) reduced by at least 67% in all cases except one (55%). However, the ability of the triphasic peptide P81 to inhibit the NP carriage in mice is not as effective as that of the biphasic peptide, with 53% and 89% significant (P <0) in serotype 2 and serotype 4, respectively. .05) demonstrated inhibition (Table 11). The nasopharyngeal carriage inhibition for serotype 6B was only 39% (P = 0.14). For these examples where biphasic and triphasic peptide inhibition was significant (P <0.05), the mean values were 80% and 60%, respectively, for the average number of Pnc bacteria colonizing the NP cavity. %Diminished.
[0163]
(Table 10. Inhibition of Pnc NP carriage by non-lipid solubilized PsaA MAP in mouse model)
[0164]
[Table 10]

^a Data represent the median number of colony forming units (cfu) / mouse (n = 8) recovered in 100 μl wash from the nasopharyngeal cavity. Mice were subcutaneously 10 ⁶ I attacked with cfu.
^b Significant difference (P <0.05) relative to control mice injected with either phosphate buffered saline or alum. Mann-Whitney rank sum test is used to calculate significant differences between groups.
(Table 11. Inhibition of Pnc NP carriage by fat-solubilized PsaA MAP in mouse model)
[0165]
[Table 11]

^a Data represent the median number of colony forming units (cfu) / mouse (n = 8) recovered in 100 μl wash from the nasopharyngeal cavity. Mice were subcutaneously 10 ⁶ I attacked with cfu.
^b Significant difference (P <0.05) relative to control mice injected with either phosphate buffered saline or alum. The Mann-Whitney rank sum test was used to calculate significant differences between groups.
Table 12. Inhibition of Pnc NP carriage by combination of PsaA MAP, biphasic peptide or triphasic peptide in mouse model
[0166]
[Table 12]

^a Data represent the median number of colony forming units (cfu) / mouse (n = 8) recovered in 100 μl wash from the nasopharyngeal cavity. Mice were subcutaneously 10 ⁶ I attacked with cfu.
^b Significant difference (P <0.05) relative to control mice injected with either phosphate buffered saline or alum. The Mann-Whitney rank sum test was used to calculate significant differences between groups.
[Brief description of the drawings]
[Figure 1]
FIG. 1A shows a biphasic peptide heterologous MAP having alternating peptide arms having the sequence of either SEQ ID NO: 5 or SEQ ID NO: 6.
FIG. 1B shows a biphasic peptide heterologous MAP with alternating peptide arms having the sequence of either SEQ ID NO: 5 or SEQ ID NO: 9.
FIG. 1C shows a triphasic peptide heterologous MAP having a first arm having the sequence of SEQ ID NO: 5, a second arm having the sequence of SEQ ID NO: 10, and a third arm having the sequence of SEQ ID NO: 9. The carboxy terminal lysine (ie, K) residue in each peptide is shared between the two arms of MAP. NLe is norleucine.
FIG. 2A
2A and 2B are diagrams showing the basic structure of non-lipidated MAP (FIG. 2A) and the basic structure of lipidated MAP (FIG. 2B). Where “B” represents three 15 amino acid based peptides shown as any of P1, P2 or P3; “J” represents a palmitoyl (palmitic acid) group, which is a 3 amino acid chain shown as U Linked to the N-terminus of the base peptide (P1, P2 and P3) via a linker (-cysteine-serine-serine-) (Tam JP, Multiple antigenic peptide system: a novel design of synthetic peptide) , JP and Kaiser, ET, New York: Alan R. Liss, Inc., 1989, pp. 3-18); P43 and P46, B = P1 (SEQ ID NO: 5); P44 and P47, B = P2 (SEQ ID NO: 6); and P45 and P48, B = P3 (SEQ ID NO: 7).
FIG. 2B
2A and 2B are diagrams showing the basic structure of non-lipidated MAP (FIG. 2A) and the basic structure of lipidated MAP (FIG. 2B). Where “B” represents three 15 amino acid based peptides shown as any of P1, P2 or P3; “J” represents a palmitoyl (palmitic acid) group, which is a 3 amino acid chain shown as U Linked to the N-terminus of the base peptide (P1, P2 and P3) via a linker (-cysteine-serine-serine-) (Tam JP, Multiple antigenic peptide system: a novel design of synthetic peptide) , JP and Kaiser, ET, New York: Alan R. Liss, Inc., 1989, pp. 3-18); P43 and P46, B = P1 (SEQ ID NO: 5); P44 and P47, B = P2 (SEQ ID NO: 6); and P45 and P48, B = P3 (SEQ ID NO: 7).
[Fig. 3]
FIG. 3 shows the basic structure of the biphasic peptides P79 and P80. Here, the base peptide is linked together by the two amino acid linker lysine-norleucine (K-NLe) to form a four-branched biphasic peptide (Srivastava N et al., Hybridoma 19: 23-31, 2000; and Tam JP (supra)). P79 consists of two units of each of the base peptides P1 and P2. In biphasic P80, P2 units are replaced with P3 units.
[Fig. 4]
FIG. 4 is a diagram showing the basic structure of the triphasic peptide P81. Here, the base peptide is linked together by the two amino acid linker lysine-norleucine (K-NLe) to form a tri-branched triphasic peptide consisting of base peptides P1, P2 and P3 (Srivastava N (previous And Tam, JP (supra)).

Claims (22)

Streptococcus pneumoniae PsaA or a multi-antigenic peptide that immunospecifically binds to a monoclonal antibody obtained in response to animal immunization using an immunogenic fragment of PsaA. The peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and immunogenic fragments of SEQ ID NOs: 5-10. The multi-antigenic peptide according to claim 1. The multi-antigenic peptide of claim 2, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof. The multi-antigenic peptide of claim 2, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 6 or an immunogenic fragment thereof. The multi-antigenic peptide of claim 2, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 7 or an immunogenic fragment thereof. The multi-antigenic peptide of claim 2, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 8 or an immunogenic fragment thereof. The multi-antigenic peptide of claim 2, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 9 or an immunogenic fragment thereof. The multi-antigenic peptide of claim 2, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 10 or an immunogenic fragment thereof. 3. The multiple antigenic peptide has at least one first arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof and at least one second arm comprising SEQ ID NO: 6 or an immunogenic fragment thereof. The multi-antigenic peptide described. 3. The multiple antigenic peptide has at least one first arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof and at least one second arm comprising SEQ ID NO: 9 or an immunogenic fragment thereof. The multi-antigenic peptide described. The multi-antigenic peptide comprises at least one first arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof; at least one second arm comprising SEQ ID NO: 6 or an immunogenic fragment thereof; and SEQ ID NO: 7 or immunity thereof. 3. A multi-antigenic peptide according to claim 2 having at least one third arm comprising an original fragment. The multi-antigenic peptide comprises at least one first arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof, at least one second arm comprising SEQ ID NO: 9 or an immunogenic fragment thereof, and SEQ ID NO: 10 or immunization thereof. 3. A multi-antigenic peptide according to claim 2 having at least one third arm comprising an original fragment. The multiple antigenic peptide according to claim 1, wherein the multiple antigenic peptide is lipid-solubilized. The multi-antigenic peptide according to claim 13, wherein the multi-antigenic peptide is lipid-solubilized with monopalmitic acid. S. in the subject. A method of conferring protective immunity against pneumoniae infection, the method comprising a plurality of immunospecific bindings to a monoclonal antibody obtained in response to immunization of an animal using Streptococcus pneumoniae PsaA or an immunogenic fragment of PsaA Administering to the subject a therapeutic composition comprising an antigenic peptide, wherein the multi-antigenic peptide comprises S. A method that is immunogenic to pneumoniae. 16. The method of claim 15, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof. 16. The method of claim 15, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 6 or an immunogenic fragment thereof. 16. The method of claim 15, wherein the multi-antigenic peptide has an arm comprising SEQ ID NO: 7 or an immunogenic fragment thereof. The multi-antigenic peptide comprises at least one first arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof; at least one second arm comprising SEQ ID NO: 6 or an immunogenic fragment thereof; and SEQ ID NO: 7 or immunity thereof. 16. A method according to claim 15 having at least one third arm comprising a protogenic fragment. The multi-antigenic peptide comprises at least one first arm comprising SEQ ID NO: 5 or an immunogenic fragment thereof, at least one second arm comprising SEQ ID NO: 9 or an immunogenic fragment thereof, and SEQ ID NO: 10 or immunization thereof. 16. A method according to claim 15 having at least one third arm comprising a protogenic fragment. 16. The method of claim 15, wherein the multi-antigenic peptide is lipid solubilized. The method of claim 21, wherein the multi-antigenic peptide is lipid solubilized with monopalmitic acid.

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